Improved wound healing compositions and treatments

ABSTRACT

This invention concerns improved methods, uses, and kits for treating chronic wounds through the administration of anti-connexin agents, particularly anti-connexin 43 antisense polynucleotides. The methods, uses, and kits of the invention are based on the surprising and unexpected discovery that chronic wounds that do not increase or decrease in size by more than a pre-determined amount during a pre-treatment phase are more amenable to successful treatment than wounds whose size varies outside the target range during the pre-treatment phase.

RELATED APPLICATIONS

This application is a 35 U.S.C. §371 National Phase Application ofInternational Application No. PCT/US14/30082, filed on Mar. 15, 2014,which claims the benefit of the filing date of U.S. application Ser. No.13/844,762, filed on Mar. 15, 2013, each of which is incorporated hereinby referenced in their entirety.

FIELD OF THE INVENTION

The inventions relate to methods of identifying and treating refractorychronic wounds that do not heal at expected rates, and dose regimens andarticles of manufacture comprising gap junction modulators useful fortreating those wounds.

BACKGROUND

The following includes information that may be useful in understandingthe present invention. It is not an admission that any of theinformation provided herein is prior art, or relevant, to the presentlydescribed or claimed inventions, or that any publication or documentthat is specifically or implicitly referenced is prior art.

In humans and other mammals, wound injury triggers an organized complexcascade of cellular and biochemical events that will in most casesresult in a healed wound. An ideally healed wound is one that restoresnormal anatomical structure, function, and appearance at the cellular,tissue, organ, and organism levels. Wound healing, whether resultingfrom trauma, microbes, or foreign materials, proceeds via a complexprocess encompassing a number of overlapping phases, includinginflammation, epithelialization, angiogenesis, and matrix deposition.Normally, these processes lead to a mature wound and a certain degree ofscar formation. Although inflammation and repair mostly occur along aprescribed course, the sensitivity of the process is dependent on thebalance of a variety of wound healing modulating factors, including, forexample, a complex network of regulatory cytokines and growth factors.

Gap junctions are cell membrane structures that facilitate directcell-cell communication. A gap junction channel is formed of twoconnexons (hemichannels), each composed of six connexin subunits. Eachhexameric connexon docks with a connexon in the opposing membrane toform a single gap junction. Gap junction channels are reported to befound throughout the body.

Connexins are a family of proteins, commonly named according to theirmolecular weight or classified on a phylogenetic basis into alpha, beta,and gamma subclasses. At least 20 human and 19 murine isoforms have beenidentified. Different tissues and cell types are reported to havecharacteristic patterns of connexin protein expression.

Antisense technology has been reported for the modulation of theexpression for genes implicated in viral, fungal, and metabolicdiseases. See, e.g., U.S. Pat. Nos. 5,166,195, 5,004,810. Antisensetechnology has also been developed to modulate connexins and treatwounds. See, e.g., U.S. Pat. Nos. 7,098,190, 7,879,811, 7,902,164,7,919,474, 8,034,789, 8,059,486, 8,063,023, 8,181,580, and 8,314,074.Peptide inhibitors of gap junctions and hemichannels have also beenreported. See, e.g., WO2006/134494, published U.S. patent applicationpublication no. 20100279921.

Despite advances in the understanding of the principles underlying thewound healing process, there remains a significant unmet need insuitable therapeutic options for chronic wound care. These inventionsaddress this continuing need.

BRIEF SUMMARY

The inventions described and claimed herein have many attributes andembodiments including, but not limited to, those set forth or describedor referenced in this summary section, which is not intended to beall-inclusive. The inventions described and claimed herein are notlimited to or by the features or embodiments identified in this summarysection, which is included for purposes of overview illustration onlyand not limitation.

This disclosure relates to methods of identifying and treatingrefractory chronic wounds, and dose regimens and articles of manufactureuseful for treating those wounds. Pharmaceutical compositions comprisingthe articles of manufacture, and useful in the methods disclosed hereincomprise effective doses of anti-connexin polynucleotides (e.g., aconnexin antisense oligodeoxynucleotide such as a single-strandedanti-connexin oligodeoxynucleotide) to connexin 26, connexin 30 and/orconnexin 43, as well as other connexins as disclosed herein.

As used herein, in one embodiment of the invention, and by way ofexample, refractory wounds are chronic wounds, or wounds that do notheal at expected rates, such as delayed-healing wounds and incompletelyhealing wounds, which do not decrease in size by more than about 30%(+30%) over a standard-of-care treatment period using, for example,compression bandaging or off-loading devices, over about two to fourweeks and which do not increase in wound size by more than 15% (−15%).This standard-of-care treatment period may also be referred to as a“run-in” or “pretreatment” period. The increase or decrease in woundsize may be referred to broadly as “surface area reduction” (alsoreferred to as SAR”). The SAR range during run-in, e.g., −15% to +30%/a,may be referred to as the run-in SAR range. Upon presentation of achronic wound for treatment, standard-of-care is provided for a two tofour week period. Estimates and/or formal measurements of the size ofthe wound upon presentation, and at the end of the run-in period areused to determine if the wound is refractory to standard of caretreatment by assessing whether it falls with a designated SAR run-inrange. In some aspects, standard of care during the run-in period ismulti-layer compression bandaging, for example. In other aspects, thestandard of care is off-loading, for example. In some embodiments, thestandard of care treatment during the run-in period is single-layercompression bandaging, or a compression stocking. Compression bandagesare typically changed or reapplied approximately once per week. In otherembodiments, the standard of care treatment during the run-in period isreduction of pressure, or offloading. Foot pressures, shock, and shearmay be reduced with appropriately fitted shoes, insoles, and socks.Total non-weight bearing using a wheelchair or crutches is anothereffective method of relieving pressure. Other off-loading standard ofcare modalities include total contact casts, removal casts, andremovable cast walkers.

In other embodiments, refractory wounds are chronic wounds, or woundsthat do not heal at expected rates, such as delayed-healing wounds andincompletely healing wounds, which do not decrease in size by more thanabout 30% (+30%) over a standard-of-care treatment period usingcompression bandaging over about two to four weeks.

In still other embodiments, refractory wounds are chronic wounds, orwounds that do not heal at expected rates, such as delayed-healingwounds and incompletely healing wounds, which do not decrease in size bymore than about 35% (+35%) over a standard-of-care treatment periodusing compression bandaging which do not increase in wound size by morethan 15% (−15%).

In further embodiments, refractory wounds are chronic wounds, or woundsthat do not heal at expected rates, such as delayed-healing wounds andincompletely healing wounds, which do not increase or decrease in sizeby more than about −5%/+30%, −10%/+30%, −15%/+30%, −20%/+30%, 25%/+30%or −30%/+30%, over a standard-of-care treatment period using compressionbandaging over about two to four weeks.

It has been surprisingly observed that the refractory chronic wounds aresusceptible to administration of pharmaceutical compositions comprisinga pharmaceutically acceptable carrier suitable for topicaladministration and, for example, from about 0.5 mg/mL to about 40 mg/mL,or by way of additional example from about 1 to 30 mg/mL, of ananti-connexin 26, anti-connexin 30 or anti-connexin 43 polynucleotide.

Accordingly, in one aspect, this disclosure relates to a method oftreating a refractory wound, the method comprising:

determining a size of a chronic wound upon initial presentation fortreatment to obtain a first size measurement or estimation;

administering standard-of-care, such as, for example, compressionbandaging and/or off-loading, to the wound;

determining the size of the wound about 2-4 weeks after administeringsaid standard-of-care to obtain a second size measurement;

determining that the second size indicator of the wound is within apredetermined range (for example, −15 to +30%/−35% to +30%) of the firstsize measurement, or that it has not healed by more a predeterminedamount (e.g., a wound SAR of more than about +30-35%) therebyidentifying a refractory wound; and

administering at or in proximity to the wound a pharmaceuticalcomposition comprising an effective amount of an anti-connexin 43polynucleotide, for example. Examples of suitable doses, dose amountsand dose concentrations and formulations are described herein.

The size of the wound may be determined by various methods known in theart, including, for example, by a measurement or reasonable estimationor approximation of any physical dimension of the wound, such as surfacearea, length of the longest diameter and/or length of the longerperpendicular bisector to the longest diameter. For deep wounds with arelatively small surface area, such as a diabetic foot ulcers, volumemay also be used as the size measurement. Other known wound measurementmethods include planimetry, wound tracing, digitizing techniques, andstereophotogrammetry, as well as simple ruler-based methods. Threecommercially available wound measurement techniques include the Visitraksystem (Smith and Nephew Healthcare, Hull, U.K.), a digital photographyand image processing system (Analyze, version 6.0; AnalyzeDirect,Lenexa. Kans.), and an elliptical measurement method using the standardformula (nab) for the calculation of the area of an ellipse.

In another aspect, this disclosure relates to methods of detecting arefractory chronic wounds susceptible to treatment by pharmaceuticalcompositions comprising, for example, from about 0.5 mg/mL to about 40mg/mL or from about 1 to 30 mg/mL of, for example, an anti-connexin 43polynucleotide, the method comprising

measuring a size indicator of a wound upon initial presentation fortreatment to obtain a first size measurement;

administering standard-of-care, such as, for example, compressionbandaging and/or off-loading, to the wound;

measuring the area of the wound about 2-4 weeks after administeringinitiating standard of care treatment to obtain a second sizemeasurement;

determining that the second size indicator of the wound is within apredetermined range of the first size measurement as described herein,thereby detecting a slowly progressing or refractory wound susceptibleto treatment by administration of a pharmaceutical compositioncomprising a therapeutically effective amount of, for example, ananti-connexin 43 polynucleotide. Other connexin targets are contemplatedas described herein.

In another aspect, this disclosure relates to kits, packages and/orarticles of manufacture useful in treating a subject having a refractorywound, comprising a receptacle containing a pharmaceutical compositioncomprising an anti-connexin 43 antisense oligodeoxynucleotide present ata therapeutically effective amount or concentration, e.g., from 1.0 to3.0 to about 30.0 to 100 milligrams per milliliter, and apharmaceutically acceptable carrier, e.g., a nonionicpolyoxyethylene-polyoxypropylene copolymer; and instructions for use ofthe compositions as described and claimed herein. Such medicamentsinclude those for the treatment of a subject as described herein.

In another aspect this invention relates to a method of detecting arefractory wound with an increased likelihood of complete closurefollowing topical administration to the wound of a compositioncomprising a nonionic polyoxyethylene-polyoxypropylene copolymer, e.g.,poloxamer 407 (at a concentration, for example, of about 15-30%, such as25-27%) and a single-stranded anti-connexin 43 antisenseoligodeoxynucleotide, for example, present at a concentration from about3.0 to about 30 milligrams per milliliter, or from about 1.0 to about100 milligrams per milliliter, for example, the method comprising:

measuring a size indicator of a wound upon initial presentation fortreatment to obtain a first size measurement;

administering standard-of-care, such as, for example, compressionbandaging and/or off-loading, to the wound;

measuring the size indicator of the wound about 2-4 weeks afterinitiating the standard of care treatment to obtain a second sizemeasurement; and

detecting that the area of the wound is within a predetermined sizerange, for example from about −15% to +30% of its size after the run-inperiod, thereby determining an increased likelihood of complete closurefollowing topical administration to the refractory wound of acomposition comprising said anti-connexin 43 antisenseoligodeoxynucleotide.

In some embodiments, the pharmaceutical composition comprises aneffective amount of a 3-30 mg/mL or 1-100 mg/mL anti-connexin 43polynucleotide or other suitable doses disclosed herein. Thepharmaceutical composition may further comprise one or morepharmaceutical carriers suitable for topical administration. In oneembodiment, the pharmaceutical composition may comprise about 20-30%nonionic polyoxyethylene-polyoxypropylene copolymer, and/or otherpharmaceutical carriers disclosed herein.

In one aspect the anti-connexin polynucleotide may be present in thepharmaceutical composition at a concentration of from about 3.0 to about30 mg/mL, or from about 1.0 to about 100 mg/mL. In some aspects, theanti-connexin polynucleotide may be present at a concentration of about3, about 5, about 10, or about 30 mg/ml, or any amount up to about 100mg/mL. In other aspects the anti-connexin polynucleotide may be presentin the pharmaceutical composition at a concentration of from about 100μM to about 5000 μM, for example. In some embodiments, a therapeuticallyeffective amount of a composition of the invention comprises apharmaceutically acceptable carrier and an anti-connexin agent such as asingle-stranded anti-connexin 43 oligodeoxynucleotide present at aconcentration from about 0.5 to about 40 mg per milliliter (mL; mg/mL)or from about 3 to about 30 mg/mL, or from about 100 μM to about 5000μM.

One or more doses may be administered to a subject having a refractorywound. In some embodiments, one or more doses of the pharmaceuticalcomposition may be repeatedly administered at appropriate or desiredintervals. In some embodiments, the pharmaceutical composition may beadministered repeatedly, for example, daily, or one to six times perweek. For example, the anti-connexin agent-containing compositions andformulations described herein can be administered once per week untilhealing is seen to be proceeding or is complete, as desired.Compositions of the invention may also be applied more frequently, forexample, two or three times/week. They may also be applied biweekly, ormonthly. The frequency of administration and dose may change over thecourse of treatment as the wound area and volume change. In addition,further application(s) can be made in the event wound healing once againbecomes stalled or delayed.

According to another aspect of the present invention, woundre-epithelialization and/or formation of granulation tissue is promoted.Methods of promoting re-epithelialization of skin wounds compriseadministering to a subject having a wound that is not healing at theexpected rate, including, for example, a delayed healing or anincompletely healing wound or a chronic wound, an anti-connexin agent,e.g., an anti-connexin polynucleotide, in an amount effective to promotere-epithelialization. Analogous methods can be used to regulateepithelial basal cell division and growth.

It has been surprisingly observed that the refractory wounds aresusceptible to administration of pharmaceutical compositions comprisingfrom 0.5 to 40 mg/mL anti-connexin polynucleotide, for example, orhigher concentrations, at or in proximity to the wound a pharmaceuticalcomposition comprising and a pharmaceutically acceptable carriersuitable for topical administration. Detecting refractory wounds mayadvantageously be used to identify patients amenable to treatment withpharmaceutical compositions comprising from 0.5 to 40 mg/mLanti-connexin polynucleotide, e.g., from 3 to 30 mg/mL of anti-connexin43 ODN, by way of example. Other suitable doses and dose concentrationsare provided herein.

In some aspects of the methods of this invention, as noted above, thesize of a patient's wound is assessed by ascertaining length, width,depth, edge circumference, volume, or surface area of the wound, orfunction thereof. Any suitable method may be used, including directmanual measurement, a laser scanner, an imaging device such as a camera,computer tablet or PDA, surface mapping, etc. The currently preferredmetric for wound size is wound surface area, preferably obtained as theproduct of wound length multiplied by wound width, wound tracing, or byplanimetry.

Size changes during the run-in period surprisingly and unexpectedly havebeen discovered to range from an increase in wound size by not more thanabout 30% to a decrease in wound size by not more than 15% during therun in period, for example, and other ranges and/or threshold amounts aspreviously noted. Preferred run-in phases range from about 7 to about 30days, preferably from about 7 days to about 21 days, even morepreferably from about 7 days to about 14 days. A 14-day run-in ispreferred.

These and other aspects of the present inventions, which are not limitedto or by the information in this Brief Summary, are provided below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C show the results from a Phase 2B clinical study using a −40%to +40% wound size change during the run in period following a 10 weektreatment period.

FIG. 1A shows the plot for % complete wound closure the patients in eachstudy arm: standard of care (SOC), vehicle alone, and low dose (1 mg) orhigh dose (1 mg) of an exemplary anti-connexin 43 polynucleotide. FIG.1B shows the plot for wound surface area reduction for patients in eachstudy arm. FIG. 1C shows that patients selected based on the −40% to+40% wound size change in the run in period did not exhibitstatistically significant responses in the context of the surface areareduction endpoint and the complete wound closure secondary endpoint.

FIG. 2 shows the plot of % complete wound closure at week 10 from are-analysis of the data from the Phase 2B study a patient subpopulationin each arm of the study that met −15% to +30% wound size changecriteria.

FIG. 3 shows the plot of surface area reduction based on the re-analysisof the data from the Phase 2B study for patient subpopulation that met−15% to +30% wound size change criteria in each arm of the study.

FIG. 4 shows that the patient subpopulation that met the −15% to +30%wound size change criteria exhibited statistically significant (P<0.05)responses in the context of the surface area reduction endpoint and thecomplete wound closure secondary endpoint.

DETAILED DESCRIPTION

Wounds that do not heal at expected rates, including slow-healingwounds, delayed-healing wounds, incompletely healing wounds, dehiscentwounds, and chronic wounds, often result in infection and can lead toamputation or death. Cell-cell communication through the gap junctionsplays pivotal roles in wound healing. It has been discovered that use ofcertain compounds, namely anti-connexin agents, including thosedescribed or referenced herein or otherwise now known or laterdeveloped, can block, inhibit, or alter cell communication to promoteclosure and healing in wounds that do not heal at expected rates,including slow-healing wounds, delayed-healing wounds, incompletelyhealing wounds, dehiscent wounds, and chronic wounds. Furthermore, asdescribed herein, it has surprisingly and unexpectedly been discoveredthat healing of refractory chronic wounds by administration of one ormore anti-connexin agents to connexin 26 (Cx26), connexin 30 (Cx30) orconnexin 43 (Cx43), for example, can be further promoted byadministering a desired anti-connexin agents to a patient whose chronicwound(s) remain within a certain size range during a standard of care(pre-treatment) run-in phase. Other connexin targets include connexin30.3 (Cx30.3), connexin 31 (Cx31), connexin 31.1 (Cx31.1), connexin 32(Cx32), connexin 37 (Cx37), connexin 40 (Cx40), and connexin 45 (Cx45),i.e., is an anti-connexin 26, 30, 30.3, 31, 31.1, 32, 37, 40, 43, or 45polynucleotide. Preferably, during a pretreatment phase of from about 1to about 30 days, preferably from about 5 days to about 20 days, evenmore preferably from about 7 days to about 14 days, the wound to betreated does not increase in size by more than about 30%, for example,or increase in size by more than about 35% as another example, orincrease in size by more than about 30% or decrease in size by more thanabout 15%, for example.

It has been surprisingly observed that the refractory wounds that do notincrease in wound size by about 30% or decrease in wound size by 15% aresusceptible to administration of pharmaceutical compositions comprisingfrom about 0.5 mg/mL to about 40 mg/mL, or from about 1 to 30 mg/mLanti-connexin polynucleotide at or in proximity to the wound apharmaceutical composition comprising and a pharmaceutically acceptablecarrier suitable for topical administration. In other embodiments,refractory wounds are chronic wounds, or wounds that do not heal atexpected rates, such as delayed-healing wounds and incompletely healingwounds, which do not decrease in size by more than about 30% (+30%) overa standard-of-care treatment period using, for example, compressionbandaging or off-loading devices over about two to four weeks. In stillother embodiments, refractory wounds are chronic wounds, or wounds thatdo not heal at expected rates, such as delayed-healing wounds andincompletely healing wounds, which do not decrease in size by more thanabout 35% (+35%) over a standard-of-care treatment period using, forexample, compression bandaging or off-loading devices, which do notincrease in wound size by more than 15% (−15%). In further embodiments,refractory wounds are chronic wounds, or wounds that do not heal atexpected rates, such as delayed-healing wounds and incompletely healingwounds, which do not increase or decrease in size by more than about−5%/+30%, −10%/+30%, −15%/+30%, −20%/+30%, 25%/+30% or −30%/+30%, over astandard-of-care treatment period using, for example, compressionbandaging or off-loading devices, over about two to four weeks. In otherembodiments, the standard of care treatment during the run-in period isreduction of pressure, or offloading. Foot pressures, shock, and shearmay be reduced with appropriately fitted shoes, insoles, and socks.Total non-weight bearing using a wheelchair or crutches is anothereffective method of relieving pressure. Other off-loading standard ofcare modalities include total contact casts, removal casts, andremovable cast walkers.

In some embodiments, a therapeutically effective amount of a compositionof the invention comprises a pharmaceutically acceptable carrier and ananti-connexin agent such as a single-stranded anti-connexin 43oligodeoxynucleotide present at a concentration from about 0.5 to about40.0 mg per milliliter (mL; mg/mL) or from about 3 to about 30 mg/mL.Preferred concentrations range from about 1.5 to about 30 milligrams permilliliter (mg/mL), about 1.5 to about 10 mg/mL, or about 3, about 5,about 10, or about 30 mg/ml. In some aspects the total dose ofanti-connexin polynucleotide administered may be about 100 μg to about30 mg.

Particularly preferred concentrations for connexin antisensepolynucleotides (e.g., single-stranded anti-connexin 43oligodeoxynucleotides) range from about 150 μM to about 10,000 μM.Viewed another way, a refractory chronic skin wound can be effectivelytreated in accordance with the invention by administering about 150 μgto about 10,000 μg of a connexin antisense polynucleotide (e.g., asingle-stranded anti-connexin 43 oligodeoxynucleotide) per squarecentimeter of wound surface area.

DEFINITIONS

As used herein, a “disorder” is any disorder, disease, or condition thatwould benefit from an agent that promotes healing of chronic or delayedhealing wounds which are refractory to standard of care treatment,and/or reduces scar formation when such wounds are treated. For example,included are wound-associated abnormalities in connection withneuropathic, ischemic, and microvascular pathology; pressure over bonyarea [tailbone (sacral), hip (trochanteric), buttocks (ischial), or heelof the foot]; reperfusion injury; and conditions associated with valvereflux etiology and related conditions.

As used herein, “subject” refers to any mammal, including humans,domestic and farm animals, and zoo, sports, or pet animals, such asdogs, horses, cats, sheep, pigs, cows, etc. The preferred mammal hereinis a human, including adults, children, and the elderly.

As used herein, “preventing” means preventing in whole or in part, orameliorating or controlling.

As used herein, a “therapeutically effective amount” in reference to thecompounds or compositions of the instant invention refers to the amountsufficient to induce a desired biological, pharmaceutical, ortherapeutic result. That result can be alleviation of the signs,symptoms, or causes of a disease or disorder or condition, or any otherdesired alteration of a biological system. In the present invention, theresult will involve the promotion and/or improvement of wound healing,including rates of wound healing and closure of wounds, in whole or inpart. Other benefits include decreases in swelling, inflammation and/orscar formation, in whole or in part.

As used herein, the term “treating” refers to both therapeutic treatmentand prophylactic or preventative measures.

As used herein, “simultaneously” is used to mean that the one or moreanti-connexin agents (e.g., an anti-connexin polynucleotide, e.g., anantisense polynucleotide) are administered concurrently, whereas theterm “in combination” is used to mean they are administered, if notsimultaneously or in physical combination, then “sequentially” within atimeframe that they both are available to act therapeutically. Thus,administration “sequentially” may permit one agent to be administeredwithin minutes (for example, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30) minutesor a matter of hours, days, weeks or months after the other providedthat one or more anti-connexin polynucleotides are concurrently presentin effective amounts. The time delay between administration of thecomponents will vary depending on the exact nature of the components,the interaction therebetween, and their respective half-lives.

As used herein, an “anti-connexin agent” decreases or inhibitsexpression of a connexin mRNA, pre-mRNA, and/or connexin protein.Anti-connexin agents include anti-connexin polynucleotides include,without limitation, antisense compounds such as antisensepolynucleotides, other polynucleotides (such as polynucleotides havingsiRNA or ribozyme functions), peptidomimetics, and other compounds thatinterfere with connexin protein activity, function, transport,localization, etc. Suitable examples of an anti-connexin polynucleotideinclude an antisense polynucleotide that targets a connexin mRNA.Accordingly, suitable anti-connexin polynucleotides include, forexample, antisense polynucleotides (e.g., Cx43 antisensepolynucleotides) that modulate expression or activity of connexins andgap junctions in selected tissues, cells, and subjects.

The term “wound dressing” refers to a dressing for topical applicationto a wound and excludes compositions suitable for systemicadministration. For example, the one or more anti-connexin agents (suchas an anticonnexin polynucleotide) may be dispersed in or on a solidsheet of wound contacting material such as a woven or nonwoven textilematerial, or may be dispersed in a layer of foam such as polyurethanefoam, or in a hydrogel such as a polyurethane hydrogel, a polyacrylatehydrogel, gelatin, carboxymethyl cellulose, pectin, alginate, and/orhyaluronic acid hydrogel, for example in a gel or ointment. In certainembodiments the one or more anti-connexin agents are dispersed in or ona biodegradable sheet material that provides sustained release of theactive ingredients into the wound, for example a sheet of freeze-driedcollagen, freeze-dried collagen/alginate mixtures (available under theRegistered Trade Mark FIBRACOL from Johnson & Johnson Medical Limited)or freeze-dried collagen/oxidized regenerated cellulose (available underthe Registered Trade Mark PROMOGRAN from Johnson & Johnson MedicalLimited).

As used herein, “wound promoting matrix” includes for example, syntheticor naturally occurring matrices such as collagen, acellular matrix,crosslinked biological scaffold molecules, tissue based bioengineeredstructural framework, biomanufactured bioprostheses, and other implantedstructures such as for example, vascular grafts suitable for cellinfiltration and proliferation useful in the promotion of wound healing.Additional suitable biomatrix material may include chemically modifiedcollagenous tissue to reduces antigenicity and immunogenicity. Othersuitable examples include collagen sheets for wound dressings,antigen-free or antigen reduced acellular matrix (Wilson, et al. (1990),Trans Am Soc Artif Intern 36:340-343), or other biomatrices that havebeen engineered to reduce the antigenic response to the xenograftmaterial. Other matrices useful in promotion of wound healing mayinclude for example, processed bovine pericardium proteins comprisinginsoluble collagen and elastin (Courtman, et al. (1994), J Biomed MaterRes 28:655-666) and other acellular tissue which may be useful forproviding a natural microenvironment for host cell migration toaccelerate tissue regeneration (Malone J M et al. (1984) J Vasc Surg1:181-91). The invention contemplates a synthetic or natural matrixcomprising one or more anti-connexin agents described herein.

As used herein, the term “wound” includes an injury to any tissue,including for example, delayed or difficult to heal wounds, and chronicwounds. Examples of wounds may include both open and closed wounds. Theterm “wound” may also include for example, injuries to the skin andsubcutaneous tissue initiated in different ways (e.g., pressure soresfrom extended bed rest and wounds induced by trauma) and with varyingcharacteristics. Wounds may be classified into one of four gradesdepending on the depth of the wound: i) Grade I wounds limited to theepithelium; ii) Grade II wounds extending into the dermis; iii) GradeIII wounds extending into the subcutaneous tissue; and iv) Grade IV (orfull-thickness wounds) wounds wherein bones are exposed (e.g., a bonypressure point such as the greater trochanter or the sacrum).

The term “partial thickness wound” refers to wounds that encompassGrades I-III. Examples of partial thickness wounds include pressuresores, venous stasis ulcers, and diabetic ulcers. The present inventioncontemplates treating all wounds of a type that do not heal at expectedrates, including, delayed-healing wounds, incompletely healing wounds,and chronic wounds.

“Wound that does not heal at the/an expected rate” means an injury toany tissue, including delayed or difficult to heal wounds (includingdelayed or incompletely healing wounds), and chronic wounds. Examples ofwounds that do not heal at the expected rate include ulcers, such asdiabetic ulcers, diabetic foot ulcers, vasculitic ulcers, arterialulcers, venous ulcers, venous stasis ulcers, pressure ulcers, decubitusulcers, infectious ulcers, trauma-induced ulcers, burn ulcers,ulcerations associated with pyoderma gangrenosum, and mixed ulcers.Other wounds that do not heal at expected rates include dehiscentwounds.

As used herein, a “delayed” or “difficult to heal” wound may include,for example, a wound that is characterized at least in part by 1) aprolonged inflammatory phase, 2) a slow forming extracellular matrix,and/or 3) a decreased rate of epithelialization or closure.

The term “chronic wound” generally refers to a wound that has nothealed. Wounds that do not heal within three months, for example, areconsidered chronic. Chronic wounds include venous ulcers, venous stasisulcers, arterial ulcers, pressure ulcers, diabetic ulcers, diabetic footulcers, vasculitic ulcers, decubitus ulcers, burn ulcers, trauma-inducedulcers, infectious ulcers, mixed ulcers, and pyoderma gangrenosum. Thechronic wound may be an arterial ulcer that comprises ulcerationsresulting from complete or partial arterial blockage. The chronic woundmay be a venous or venous stasis ulcer that comprises ulcerationsresulting from a malfunction of the venous valve and the associatedvascular disease. In certain embodiments a method of treating a chronicwound is provided where the chronic wound is characterized by one ormore of the following AHCPR stages of pressure ulceration: stage 1,stage 2, stage 3, and/or stage 4.

As used herein, chronic wound may refer to, for example, a wound that ischaracterized at least in part by one or more of (1) a chronicself-perpetuating state of wound inflammation, (2) a deficient anddefective wound extracellular matrix, (3) poorly responding (senescent)wound cells especially fibroblasts, limiting extracellular matrixproduction, and/or (4) failure of re-epithelialization due in part tolack of the necessary extracellular matrixorchestration and lack ofscaffold for migration. Chronic wounds may also be characterized by 1)prolonged inflammation and proteolytic activity leading to ulcerativelesions, including for example, diabetic, pressure (decubitous), venous,and arterial ulcers; 2) progressive deposition of matrix in the affectedarea, 3) longer repair times, 4) less wound contraction, 5) slowerre-epithelialization, and 6) increased thickness of granulation tissue.

The term “refractory” chronic wound or “refractory” wound refers towounds that do not heal at expected rates, such as delayed-healingwounds, incompletely healing wounds, and chronic wounds, and which donot, for example, increase in wound size by more than about 30% (arepartially refractory to administration of standard of care) and which donot decrease in wound size by more than 15% (−15%) (are totallyrefractory to administration of standard of care) during a two to fourweek run-in period. Following presentation for treatment of a wound thatdoes not heal at the expected rate, the wound is pre-treated usingstandard of care treatment using, for example, compression bandaging oroff-loading devices. In some embodiments the refractory wounds arewounds that do not heal at expected rates and which do not increase inwound size by more than about 30% and do not decrease in size by morethan about 20%, 25%, 30%, or 35% during the run-in period. In otherembodiments, refractory wounds are chronic wounds, or wounds that do notheal at expected rates, such as delayed-healing wounds and incompletelyhealing wounds, which do not decrease in size by more than about 30%(+30%) over a standard-of-care treatment period using, for example,compression bandaging or off-loading devices, over about two to fourweeks. In still other embodiments, refractory wounds are chronic wounds,or wounds that do not heal at expected rates, such as delayed-healingwounds and incompletely healing wounds, which do not decrease in size bymore than about 35% (+35%) over a standard-of-care treatment periodusing, for example, compression bandaging or off-loading devices, whichdo not increase in wound size by more than 15% (−15%). In furtherembodiments, refractory wounds are chronic wounds, or wounds that do notheal at expected rates, such as delayed-healing wounds and incompletelyhealing wounds, which do not increase or decrease in size by more thanabout −5%/+30%, −10%/+30%, −15%/+30%, −200/+30%, 25%/+30% or −30/+30%,over a standard-of-care treatment period using, for example, compressionbandaging or off-loading devices, over about two to four weeks. In someaspects of this disclosure, the refractory wound may be, for example, arefractory skin ulcer, such as a venous leg ulcer, or diabetic footulcer. In yet another aspect, the invention includes methods fortreating a subject having or suspected of having any diseases,disorders, and/or conditions characterized in whole or in part by achronic wound or delayed or incomplete wound healing, or other woundthat does not heal at an expected rate. In some embodiments, the patienthas a diabetic ulcer, a diabetic foot ulcer, a vasculitic ulcer, avenous ulcer, a venous stasis ulcer, an arterial ulcer, a pressureulcer, a decubitus ulcer, an infectious ulcer, a trauma-induced ulcer, aburn ulcer, ulcerations associated with pyoderma gangrenosum, or a mixedulcer or ulcers.

In the context of the instant inventions, the anti-connexin agents arepreferably administered topically (at and/or around the site to betreated). Suitably, the anti-connexin agents, e.g., anti-connexinantisense polynucleotides are combined with a pharmaceuticallyacceptable carrier, vehicle or diluent to provide a pharmaceuticalcomposition.

Exemplary chronic wounds may include “pressure ulcers.” Exemplarypressure ulcers may be classified into 4 stages based on AHCPR (Agencyfor Health Care Policy and Research, U.S. Department of Health and HumanServices) guidelines. A stage I pressure ulcer is an observable pressurerelated alteration of intact skin whose indicators as compared to theadjacent or opposite area on the body may include changes in one or moreof the following: skin temperature (warmth or coolness), tissueconsistency (firm or boggy feel) and/or sensation (pain, itching). Theulcer appears as a defined area of persistent redness in lightlypigmented skin, whereas in darker skin tones, the ulcer may appear withpersistent red, blue, or purple hues. Stage 1 ulceration may includenonblanchable erythema of intact skin and the heralding lesion of skinulceration. In individuals with darker skin, discoloration of the skin,warmth, edema, induration, or hardness may also be indicators of stage 1ulceration. Stage 2 ulceration may be characterized by partial thicknessskin loss involving epidermis, dermis, or both. The ulcer is superficialand presents clinically as an abrasion, blister, or shallow crater.Stage 3 ulceration may be characterized by full thickness skin lossinvolving damage to or necrosis of subcutaneous tissue that may extenddown to, but not through, underlying fascia. The ulcer presentsclinically as a deep crater with or without undermining of adjacenttissue. Stage 4 ulceration may be characterized by full thickness skinloss with extensive destruction, tissue necrosis, or damage to muscle,bone, or supporting structures (e.g., tendon, joint capsule). In certainembodiments a method of treating a chronic wound is provided where thechronic wound is characterized by one or more of the following AHCPRstages of pressure ulceration: stage 1, stage 2, stage 3, and/or stage4.

Exemplary chronic wounds may also include “decubitus ulcers.” Exemplarydecubitus ulcers may arise as a result of prolonged and unrelievedpressure over a bony prominence that leads to ischemia. The wound tendsto occur in patients who are unable to reposition themselves to off-loadweight, such as paralyzed, unconscious, or severely debilitated persons.As defined by the U.S. Department of Health and Human Services, themajor preventive measures include identification of high-risk patients;frequent assessment; and prophylactic measures such as scheduledrepositioning, appropriate pressure-relief bedding, moisture barriers,and adequate nutritional status. Treatment options may include forexample, pressure relief, surgical and enzymatic debridement, moistwound care, and control of the bacterial load. In certain embodiments amethod of treating a chronic wound is provided wherein the chronic woundis characterized by decubitus ulcer or ulceration, which results fromprolonged, unrelieved pressure over a bony prominence that leads toischemia.

Chronic wounds may also include “arterial ulcers.” Chronic arterialulcers are generally understood to be ulcerations that accompanyarteriosclerotic and hypertensive cardiovascular disease. They arepainful, sharply marginated, and often found on the lateral lowerextremities and toes. Arterial ulcers may be characterized by completeor partial arterial blockage, which may lead to tissue necrosis and/orulceration. Signs of arterial ulcer may include, for example,pulselessness of the extremity; painful ulceration; small, punctateulcers that are usually well circumscribed; cool or cold skin; delayedcapillary return time (briefly push on the end of the toe and release,normal color should return to the toe in about 3 seconds or less);atrophic appearing skin (for example, shiny, thin, dry); and loss ofdigital and pedal hair. In certain embodiments a method of treating achronic wound is provided wherein the chronic wound is characterized byarterial ulcers or ulcerations due to complete or partial arterialblockage.

Exemplary chronic wounds may include “venous ulcers.” Exemplary venousulcers are the most common type of ulcer affecting the lower extremitiesand may be characterized by malfunction of the venous valve. The normalvein has valves that prevent the backflow of blood. When these valvesbecome incompetent, the backflow of venous blood causes venouscongestion. Hemoglobin from the red blood cells escapes and leaks intothe extravascular space, causing the brownish discoloration commonlynoted. It has been shown that the transcutaneous oxygen pressure of theskin surrounding a venous ulcer is decreased, suggesting that there areforces obstructing the normal vascularity of the area. Lymphaticdrainage and flow also plays a role in these ulcers. The venous ulcermay appear near the medial malleolus and usually occurs in combinationwith an edematous and indurated lower extremity; it may be shallow, nottoo painful and may present with a weeping discharge from the affectedsite. In certain embodiments a method of treating a chronic wound isprovided wherein the chronic wound is characterized by venous ulcers orulcerations due to malfunction of the venous valve and the associatedvascular disease. In certain embodiments a method of treating a chronicwound is provided wherein the chronic wound is characterized by arterialulcers or ulcerations due to complete or partial arterial blockage.

Exemplary chronic wounds may include “venous stasis ulcers.” Stasisulcers are lesions associated with venous insufficiency are morecommonly present over the medial malleolus, usually with pitting edema,varicosities, mottled pigmentation, erythema, and nonpalpable petechiaeand purpura. The stasis dermatitis and ulcers are generally pruriticrather than painful. Exemplary venous stasis ulcers may be characterizedby chronic passive venous congestion of the lower extremities results inlocal hypoxia. One possible mechanism of pathogenesis of these woundsincludes the impediment of oxygen diffusion into the tissue across thickperivascular fibrin cuffs. Another mechanism is that macromoleculesleaking into the perivascular tissue trap growth factors needed for themaintenance of skin integrity. Additionally, the flow of large whiteblood cells slows due to venous congestion, occluding capillaries,becoming activated, and damaging the vascular endothelium to predisposeto ulcer formation. In certain embodiments a method of treating achronic wound is provided wherein the chronic wound is characterized byvenous ulcers or ulcerations due to malfunction of the venous valve andthe associated vascular disease. In certain embodiments a method oftreating a chronic wound is provided wherein the chronic wound ischaracterized by venous stasis ulcers or ulcerations due to chronicpassive venous congestion of the lower extremities and/or the resultinglocal hypoxia.

Exemplary chronic wounds may include “diabetic ulcers.” Diabeticpatients are prone to ulcerations, including foot ulcerations, due toboth neurologic and vascular complications. Peripheral neuropathy cancause altered or complete loss of sensation in the foot and/or leg.Diabetic patients with advanced neuropathy lose all ability forsharp-dull discrimination. Any cuts or trauma to the foot may gocompletely unnoticed for days or weeks in a patient with neuropathy. Itis not uncommon to have a patient with neuropathy notice that the ulcer“just appeared” when, in fact, the ulcer has been present for quite sometime. For patients of neuropathy, strict glucose control has been shownto slow the progression of the disease. Charcot foot deformity may alsooccur as a result of decreased sensation. People with “normal” feelingin their feet have the ability to sense automatically when too muchpressure is being placed on an area of the foot. Once identified, ourbodies instinctively shift position to relieve this stress. A patientwith advanced neuropathy loses this ability to sense the sustainedpressure insult, as a result, tissue ischemia and necrosis may occurleading to for example, plantar ulcerations. Additionally,microfractures in the bones of the foot, if unnoticed and untreated, mayresult in disfigurement, chronic swelling and additional bonyprominences. Microvascular disease is one of the significantcomplications for diabetics, which may also lead to ulcerations. Incertain embodiments a method of treating a chronic wound is providedwherein the chronic wound is characterized by diabetic foot ulcersand/or ulcerations due to both neurologic and vascular complications ofdiabetes.

Exemplary chronic wounds can include “traumatic ulcers.” Formation oftraumatic ulcers may occur as a result of traumatic injuries to thebody. These injuries include, for example, compromises to the arterial,venous or lymphatic systems; changes to the bony architecture of theskeleton; loss of tissue layers-epidermis, dermis, subcutaneous softtissue, muscle or bone; damage to body parts or organs and loss of bodyparts or organs. In certain embodiments, a method of treating a chronicwound is provided wherein the chronic wound is characterized byulcerations associated with traumatic injuries to the body.

Exemplary chronic wounds can include “burn ulcers”, including firstdegree burn (i.e. superficial, reddened area of skin); second degreeburn (a blistered injury site which may heal spontaneously after theblister fluid has been removed); third degree burn (burn through theentire skin and usually require surgical intervention for woundhealing); scalding (may occur from scalding hot water, grease orradiator fluid); thermal (may occur from flames, usually deep burns);chemical (may come from acid and alkali, usually deep burns); electrical(either low voltage around a house or high voltage at work); explosionflash (usually superficial injuries); and contact burns (usually deepand may occur from muffler tail pipes, hot irons and stoves). In certainembodiments, a method of treating a chronic wound is provided whereinthe chronic wound is characterized by ulcerations associated with burninjuries to the body.

Exemplary chronic wounds can include “vasculitic ulcers.” Vasculiticulcers also occur on the lower extremities and are painful, sharplymarginated lesions, which may have associated palpable purpuras andhemorrhagic bullae. The collagen diseases, septicemias, and a variety ofhematological disorders (e.g., thrombocytopenia, dysproteinemia) may bethe cause of this severe, acute condition.

Exemplary chronic wounds can include pyoderma gangrenosum. Pyodermagangrenosum occurs as single or multiple, very tender ulcers of thelower legs. A deep red to purple, undermined border surrounds thepurulent central defect. Biopsy typically fails to reveal a vasculitis.In half the patients it is associated with a systemic disease such asulcerative colitis, regional ileitis, or leukemia. In certainembodiments, a method of treating a chronic wound is provided whereinthe chronic wound is characterized by ulcerations associated withpyoderma gangrenosum.

Exemplary chronic wounds can include infectious ulcers. Infectiousulcers follow direct innoculation with a variety of organisms and may beassociated with significant regional adenopathy. Mycobacteria infection,anthrax, diphtheria, blastomyosis, sporotrichosis, tularemia, andcat-scratch fever are examples. The genital ulcers of primary syphilisare typically nontender with a clean, firm base. Those of chancroid andgranuloma inguinale tend to be ragged, dirty, and more extravagantlesions. In certain embodiments, a method of treating a chronic wound isprovided wherein the chronic wound is characterized by ulcerationsassociated with infection.

As used herein, the term “dehiscent wound” refers to a wound, usually asurgical wound, which has ruptured or split open. In certainembodiments, a method of treating a wound that does not heal at theexpected rate is provided wherein the wound is characterized bydehiscence.

Anti-Connexin Agents Anti-Connexin Polynucleotides

Anti-connexin polynucleotides include connexin antisense polynucleotidesas well as polynucleotides which have functionalities that enable themto downregulate connexin expression (for example, by downregulation ofmRNA transcription or translation). In the case of downregulation, thishas the effect of reducing direct cell-cell communication by gapjunctions at the site at which connexin expression is down-regulated.

The inventions generally relate to the use of an anti-connexin agent,preferably an anti-connexin polynucleotide, including, for example, ananti-connexin oligodeoxynucleotide (ODN), directed to a messenger RNA(mRNA) or precursor thereof that codes a connexin protein.Representative anti-connexin polynucleotides used in the methods andarticles of manufacture of this disclosure include connexin antisensepolynucleotides, as well as RNAi polynucleotides, siRNA polynucleotides,shRNA polynucleotides, ribozymes, DNAzymes, and other anti-connexinpolynucleotides that target a connexin messenger RNA (mRNA) or precursorthereof. Other anti-connexin agents within the scope of the inventioninclude peptidomimetics and connexin phosphorylation agents.

In the context of anti-connexin polynucleotides, such molecules arepreferably single-stranded polynucleotides, although under physiologicalconditions all or portions of such molecules may include one or morepartially or completely double-stranded regions. Such polynucleotidesinclude those having modified and/or unmodified backbone, and can beproduced recombinantly or synthetic chemistry.

In another embodiment, the anti-connexin polynucleotide may be ananti-connexin 43, anti-connexin 26 and anti-connexin 30 polynucleotides,for example, an ODN, such as a single-stranded anti-connexinoligodeoxynucleotide to connexin 26, connexin 30 or connexin 43. In someembodiments the anti-connexin polynucleotide is an anti-connexin 43polynucleotide. In one embodiment, the anti-connexin polynucleotide is aconnexin 43 antisense oligodeooxynucleotide such as a single-strandedanti-connexin oligodeoxynucleotide to connexin.

In certain other embodiments, the anti-connexin agent is ananti-connexin polynucleotide that targets a connexin mRNA or precursorthereof (i.e., pre-mRNA), particularly an mRNA or pre-mRNA that codesfor connexin 43 (Cx43), connexin 26 (Cx26), connexin 37 (Cx37), connexin30 (Cx30), connexin 30.3 (Cx30.3), connexin 31 (Cx31), connexin 31.1(Cx31.1), or connexin 32 (Cx32), connexin 40 (Cx40), and connexin 45(Cx45), i.e., is an anti-connexin 43, 26, 37, 30, 30.3, 31, 31.1, 32, 40or 45 polynucleotide.

Particularly preferred anti-connexin polynucleotides includeanti-connexin oligodeoxynucleotides such as anti-connexin 43oligodeoxynucleotides. Preferred anti-connexin polynucleotides containfrom about 18 to about 32 polynucleotides.

Accordingly, in another aspect, the invention provides formulationscomprising at least one anti-connexin agent, e.g., a connexin antisensepolynucleotide, together with a pharmaceutically acceptable carrier orvehicle. In one preferred form, such formulations contain a connexinantisense polynucleotide that targets a single connexin mRNA species,most preferably, connexin 43 mRNA or pre-mRNA. Alternatively, theformulation can contain an anti-connexin agent, e.g., a connexinantisense polynucleotide, that targets more than one connexin mRNAspecies, e.g., an mRNA species that codes for Cx43 as well as an mRNAspecies that codes for Cx30, Cx26, Cx37, Cx31.1, or Cx32 and others asnoted herein.

According to one aspect, the downregulation of connexin expression maybe based generally upon the antisense approach using antisensepolynucleotides (such as DNA or RNA polynucleotides), and moreparticularly upon the use of antisense oligodeoxynucleotides (ODN).These polynucleotides (e.g., ODN) target the connexin protein (s) to bedown-regulated. Typically the polynucleotides are single stranded, butmay be double stranded.

The antisense polynucleotide may inhibit transcription and/ortranslation of a connexin. Preferably the polynucleotide is a specificinhibitor of transcription and/or translation from the connexin gene ormRNA, and does not inhibit transcription and/or translation from othergenes or mRNAs. The product may bind to the connexin gene or mRNA either(i) 5′ to the coding sequence, and/or (ii) to the coding sequence,and/or (iii) 3′ to the coding sequence.

The antisense polynucleotide is generally antisense to a connexin mRNA.Such a polynucleotide may be capable of hybridizing to the connexin mRNAand may thus inhibit the expression of connexin by interfering with oneor more aspects of connexin mRNA metabolism including transcription,mRNA processing, mRNA transport from the nucleus, translation or mRNAdegradation. The antisense polynucleotide typically hybridizes to theconnexin mRNA to form a duplex which can cause direct inhibition oftranslation and/or destabilization of the mRNA. Such a duplex may besusceptible to degradation by nucleases.

The antisense polynucleotide may hybridize to all or part of theconnexin mRNA. Typically the antisense polynucleotide hybridizes to theribosome binding region or the coding region of the connexin mRNA. Thepolynucleotide may be complementary to all of or a region of theconnexin mRNA. For example, the polynucleotide may be the exactcomplement of all or a part of connexin mRNA. However, absolutecomplementarity is not required and polynucleotides which havesufficient complementarity to form a duplex having a melting temperatureof greater than about 20° C., 30° C., or 40° C. under physiologicalconditions are particularly suitable for use in the present invention.

Thus the polynucleotide is typically a homologue of a sequencecomplementary to the mRNA. The polynucleotide may be a polynucleotidewhich hybridizes to the connexin mRNA under conditions of medium to highstringency such as 0.03M sodium chloride and 0.03M sodium citrate atfrom about 50° C. to about 60° C.

For certain aspects, suitable polynucleotides are typically from about 6to 40 nucleotides in length. Preferably a polynucleotide may be fromabout 12 to about 35 nucleotides in length, or alternatively from about12 to about 20 nucleotides in length or more preferably from about 18 toabout 32 nucleotides in length. According to an alternative aspect, thepolynucleotide may be at least 40 nucleotides in length.

The inventions include pharmaceutical compositions comprising (a) atherapeutically effect amount of a pharmaceutically acceptableanti-connexin agent (e.g., an anti-connexin 43 oligodeoxynucleotide) and(b) a pharmaceutically acceptable carrier or diluent. Therapeuticallyeffective doses and dose concentrations are described herein. In someembodiments, the compositions or treatment regimens of the inventioninclude compositions that include multiple anti-connexin agent species(e.g., two or more anti-connexin polynucleotide species wherein eachspecies targets a different connexin mRNA or pre-mRNA species; ananti-connexin polynucleotide that can target two or more differentconnexin mRNA or pre-mRNA species because of conserved sequence identityover at least a portion of the different connexin mRNA or pre-mRNAspecies targeted by the anti-connexin polynucleotide; etc.).

The anti-connexin agents of this invention may include those agents orcompounds capable of inducing phosphorylation on connexin amino acidresidues in order to induce gap junction or hemichannel closure.Exemplary sites of phosphorylation include one or more of a tyrosine,serine or threonine residues on the connexin protein. In certainembodiments, modulation of phosphorylation may occur on one or moreresidues on one or more connexin proteins. Exemplary gap junctionphosphorylating agents are well known in the art and may include, forexample, c-Src tyrosine kinase or other G protein-coupled receptoragonists. See Giepmans B, J. Biol. Chem., Vol. 276, Issue 11, 8544-8549,Mar. 16, 2001. In one embodiment, modulation of phosphorylation on oneor more of these residues impacts hemichannel function, particularly byclosing the hemichannel. In another embodiment, modulation ofphosphorylation on one or more of these residues impacts gap junctionfunction, particularly by closing the gap junction. Gap junctionphosphorylating agents that target the closure of connexin 43 gapjunctions and hemichannels are preferred.

Still other anti-connexin agents include connexin carboxy-terminalpolypeptides. See Gourdie et al., WO2006/069181.

In certain another aspect, gap junction modifying agent may include, forexample, aliphatic alcohols; octanol; heptanol; anesthetics (e.g.halothane), ethrane, fluothane, propofol and thiopental; anandamide;arylaminobenzoate (FFA: flufenamic acid and similar derivatives that arelipophilic); carbenoxolone; Chalcone: (2′,5′-dihydroxychalcone); CHFs(Chlorohydroxyfuranones); CMCF(3-chloro-4-(chloromethyl)-5-hydroxy-2(5H)-furanone); dexamethasone;doxorubicin (and other anthraquinone derivatives); eicosanoidthromboxane A(2) (TXA(2)) mimetics; NO (nitric oxide); Fatty acids (e.g.arachidonic acid, oleic acid and lipoxygenase metabolites; Fenamates(flufenamic (FFA), niflumic (NFA) and meclofenamic acids (MFA));Genistein; glycyrrhetinic acid (GA):18a-glycyrrhetinic acid and18-beta-glycyrrhetinic acid, and derivatives thereof; lindane;lysophosphatidic acid; mefloquine; menadione;2-Methyl-1,4-naphthoquinone, vitamin K(3); nafenopin; okadaic acid;oleamide; oleic acid; PH, gating by intracellular acidification; e.g.,acidifying agents; polyunsaturated fatty acids; fatty acid GJICinhibitors (e.g., oleic and arachidonic acids); quinidine; quinine; alltrans-retinoic acid; and tamoxifen.

The anti-connexin agents of the invention, particularly anti-connexinpolynucleotides such as connexin antisense polynucleotides (e.g., aconnexin antisense oligodeooxynucleotide such as a single-strandedanti-connexin 43 oligodeoxynucleotide), can be used to effect treatmentof a chronic skin wound by administering to such a wound a compositionthat comprises about 0.001 milligram (mg) to about 10, 1, 0.1, or 0.01mg of the anti-connexin agent per kilogram (kg) body weight in apharmaceutically acceptable carrier. Preferred amounts of theanti-connexin agent (e.g., a single-stranded anti-connexin 43oligodeoxynucleotide) in such a composition include about 0.01 mg toabout 10 mg per kg body weight (mg/kg), about 0.01 mg to about 10 mg/kg,and about 0.5 to about 1.0 mg/kg. The dosage may also be about 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0,8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0,20.0, 21.0, 22.0, 23.0, 24.0, 25.0, 26.0, 27.0, 28.0, 29.0, 30.0, 31.0,32.0, 33.0, 34.0, 35.0, 36.0, 37.0, 38.0, 39.0, 40.0, 41.0, 42.0, 43.0,44.0, 45.0, 46.0, 47.0, 48.0, 49.0, 50.0, 52.5, 55.0, 57.5, 60.0, 62.5,65.0, 67.5, 70.0, 72.5, 75.0, 77.5, 80.0, 82.5, 85.0, 87.5, 90.0, 92.5,95.0, 97.5, or about 100.0 mg per kg body weight, or any range orsubrange between any two of the recited doses, or any dose fallingwithin the range of from about 0.1 to about 100 mg per kg body weight.

In some embodiments, one or more doses of the pharmaceutical compositionmay be administered at appropriate intervals. In some aspects, the totaldose (weight) of anti-connexin polynucleotide administered to therefractory wound may be about 100 μg to about 1 mg. In some aspects, thetotal dose (w) of anti-connexin polynucleotide administered may 100 μg,200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, 1 mg, 2mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, or about 30 mg, or anyamount ranging between any two of those doses. In other embodiments, thedoses will be about 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0,18.0, 19.0, 20.0, 21.0, 22.0, 23.0, 24.0, 25.0, 26.0, 27.0, 28.0, 29.0,30.0, 31.0, 32.0, 33.0, 34.0, 35.0, 36.0, 37.0, 38.0, 39.0, 40.0, 41.0,42.0, 43.0, 44.0, 45.0, 46.0, 47.0, 48.0, 49.0, 50.0, 52.5, 55.0, 57.5,60.0, 62.5, 65.0, 67.5, 70.0, 72.5, 75.0, 77.5, 80.0, 82.5, 85.0, 87.5,90.0, 92.5, 95.0, 97.5, 100.0, 105, 110, 115, 120, 125, 130, 135, 140,145, 150, 155, 160, 65, 170, 175, 180, 185, 190, 195, 200, 210, 220,230, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370,380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or about 500milligrams per square centimeter, or any range or subrange between anytwo of the recited doses, or any dose falling within the range of about1.0 to about 500 milligrams per square centimeter.

In some aspects the dose of anti-connexin polynucleotide is administeredin a volume of about 25 ul to about 3 ml. Preferred dose volumes forcompositions that include a pharmaceutically acceptable carrier and from0.5 to about 1.0 mg of an anti-connexin agent (e.g., a single-strandedanti-connexin 43 oligodeoxynucleotide) range from about 25-100microliter (μL), from about 100-200 μL, from about 200-500 μL, or fromabout 500-1000 μL.

In other embodiments, the anti-connexin agent is applied at about a 20μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 10-200 μM,200-300 μM, 300-400 μM, 400-500 μM, 500-600 μM, 600-700 μM, 700-800 μM,800-900 μM, 900-1000 or 1000-1500 μM, or 1500 μM-2000 μM, 2000 μM-3000μM, 3000 μM-4000 μM, 4000 μM-5000 μM, 5000 μM-6000 μM, 6000 μM-7000 μM,7000 μM-8000 μM, 8000 μM-9000 μM, 9000 μM-10,000 μM, 10,000 μM-11,000μM, 11,000 μM-12,000 μM, 12,000 μM-13,000 μM, 13,000 μM-14,000 μM,14,000 μM-15,000 μM, 15,000 μM-20,000 μM, 20,000 μM-30,000 μM, 30,000μM-50,000 μM, or greater, or any range or subrange between any two ofthe recited doses, or any dose falling within the range of from about 20μM to about 50,000 μM.

The connexin protein or proteins targeted by the polynucleotide will bedependent upon the site at which downregulation is to be effected. Thisreflects the non-uniform make-up of gap junction(s) at different sitesthroughout the body in terms of connexin sub-unit composition. Theconnexin is a connexin that naturally occurs in a human or animal in oneaspect or naturally occurs in the tissue in which connexin expression oractivity is to be decreased. The connexin gene (including codingsequence) generally has homology or nucleotide sequence identity withthe coding sequence of one or more of the specific connexins mentionedherein. The connexin is typically an a or P connexin. Preferably theconnexin is an a connexin and is expressed in the tissue to be treated.

Some connexin proteins are, however, more ubiquitous than others interms of distribution in tissue. One of the most widespread is connexin43. Polynucleotides targeted to connexin 43 are particularly suitablefor use in the present invention. In other aspects other connexins aretargeted. In one preferred aspect, the antisense polynucleotides aretargeted to the mRNA of one connexin protein only. Most preferably, thisconnexin protein is connexin 43. In another aspect, connexin protein isconnexin Cx26, Cx30, Cx30.3, Cx31, Cx31.1, Cx32, Cx37, Cx40, or Cx45. Inother aspects, the connexin protein is connexin 26 or 30.

It is also contemplated that polynucleotides targeted to separateconnexin proteins be used in combination (for example 1, 2, 3, 4, ormore different connexins may be targeted). For example, polynucleotidestargeted to connexin 43, and one or more other members of the connexinfamily (such as connexin Cx26, Cx30, Cx30.3, Cx31, Cx31.1, Cx32, Cx37,Cx40, or Cx45 can be used in combination.

Alternatively, the anticonnexin polynucleotides may be part ofcompositions that may comprise polynucleotides to more than one connexinprotein. Preferably, one of the connexin proteins to whichpolynucleotides are directed is connexin 43. Other connexin proteins towhich oligthe polynucleotides are directed may include, for example,connexin Cx26, Cx30, Cx30.3, Cx31, Cx31.1, Cx32, Cx37, Cx40, or Cx45.

Individual anti-connexin agents may be specific to a particularconnexin, or may target 1, 2, 3, or more different connexins. Specificpolynucleotides will in some embodiments target sequences in thetargeted connexin gene, mRNA, or pre-mRNA that are not conserved betweenconnexins, whereas multi-specific polynucleotides will target conservedsequences for various connexins.

The polynucleotides for use in the invention may suitably be unmodifiedphosphodiester oligomers. Such oligodeoxynucleotides may vary in length.A 30-mer polynucleotide has been found to be particularly suitable.

Many aspects of the invention are described with reference tooligodeoxynucleotides. However, it is understood that other suitablepolynucleotides (such as RNA polynucleotides) and anti-connexin agentsmay be used in these aspects.

The anticonnexin polynucleotides may be chemically modified. This mayenhance their resistance to nucleases and may enhance their ability toenter cells. For example, phosphorothioate oligonucleotides may be used.Other deoxynucleotide analogs include methylphosphonates,phosphoramidates, phosphorodithioates, N3′P5′-phosphoramidates andoligoribonucleotide phosphorothioates and their 2′-O-alkyl analogs and2′-O-methylribonucleotide methylphosphonates. Alternatively, mixedbackbone oligonucleotides (“MBOs”) may be used. MBOs contain segments ofphosphothioate oligodeoxynucleotides and appropriately placed segmentsof modified oligodeoxy- or oligoribonucleotides. MBOs have segments ofphosphorothioate linkages and other segments of other modifiedoligonucleotides, such as methylphosphonate, which is non-ionic, andvery resistant to nucleases or 2′-O-alkyloligoribonucleotides. Methodsof preparing modified backbone and mixed backbone oligonucleotides areknown in the art.

The precise sequence of representative preferred antisensepolynucleotides used in the invention will depend upon the targetconnexin protein. As described, suitable connexin antisensepolynucleotides can include polynucleotides such asoligodeoxynucleotides.

Suitable polynucleotides for the preparation of the combinedpolynucleotide compositions described herein include for example,polynucleotides to connexin 43 and polynucleotides for connexins 26, 30,31.1, 32, and 37.

Although the precise sequence of the anticonnexin polynucleotide used inthe invention will depend upon the target connexin protein, for connexin43, antisense polynucleotides having the following sequences have beenfound to be particularly suitable:

SEQ. ID. NO: 1: 5′-GTA ATT GCG GCA AGA AGA ATT GTT TCT GTC-3′SEQ. ID. NO: 2: 5′-GTA ATT GCG GCA GGA GGA ATT GTT TCT GTC-3′SEQ. ID. NO: 3: 5′-GGC AAG AGA CAC CAA AGA CAC TAC CAG CAT-3′SEQ. ID. NO: 7: 5′-GAC AGA AAC AAT TCC TCC TGC CGC ATT TAC-3′

Suitable antisense polynucleotides for connexins 26, 31.1, and 32 havethe following sequences:

SEQ. ID. NO: 4 (Cx26): 5′-TCC TGA GCA ATA CCT AAC GAA CAA ATA-3′SEQ. ID. NO: 5 (Cx31.1): 5′-CGT CCG AGC CCA GAA AGA TGA GGT C-3′SEQ. ID. NO: 6 (Cx32): 5′-TTT CTT TTC TAT GTG CTG TTG GTG A-3′

Polynucleotides, including ODN's, directed to connexin proteins can beselected in terms of their nucleotide sequence by any convenient, andconventional, approach. For example, the computer programs MacVector andOligoTech (from Oligos etc. Eugene, Oreg., USA) can be used. Onceselected, the ODN's can be synthesized using a DNA synthesizer.

Polynucleotide Homologues

Homology, homologues, and nucleotide sequence identity are describedherein (for example, the polynucleotide may be a homologue of acomplement to a sequence in connexin mRNA). Such a polynucleotidetypically has at least about 70% nucleotide sequence identity,preferably at least about 80%, at least about 90%, at least about 95%,at least about 97% or at least about 99% sequence identity with therelevant sequence, for example, over a region of at least about 15, atleast about 20, at least about 40, at least about 100 more contiguousnucleotides (of the homologous sequence). Homology or sequence identitymay be calculated based on any method in the art.

For example, the BLAST algorithm performs a statistical analysis of thesimilarity between two sequences; see, e.g., Karlin and Altschul (1993),Proc. Natl. Acad. Sci. USA 90: 5873-5787. One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a sequence is considered similar to another sequence if thesmallest sum probability in comparison of the first sequence to a secondsequence is less than about 1, preferably less than about 0.1, morepreferably less than about 0.01, and most preferably less than about0.001.

The homologous sequence typically differs from the relevant sequence byat least about (or by no more than about) 2, 5, 10, 15, 20, or moremutations (which may be substitutions, deletions or insertions). Thesemutations may be measured across any of the regions mentioned above inrelation to calculating sequence identity.

The homologous sequence typically hybridizes selectively to the originalsequence at a level significantly above background. Selectivehybridization is typically achieved using conditions of medium to highstringency (for example 0.03M sodium chloride and 0.03M sodium citrateat from about 50° C. to about 60° C.). However, such hybridization maybe carried out under any suitable conditions known in the art (seeSambrook, et al. (1989), Molecular Cloning: A Laboratory Manual). Forexample, if high stringency is required, suitable conditions include0.2×SSC at 60° C. If lower stringency is required, suitable conditionsinclude 2×SSC at 60° C.

Dosage Forms and Formulations and Administration

The anti-connexin agents of the invention of the may be administered toa subject in need of treatment, such as a subject with any of the woundsmentioned herein. The condition of the subject can thus be improved. Theanti-connexin agent may be used in the treatment of the subject's bodyby therapy. They may be used in the manufacture of a medicament to treatany of the wounds mentioned herein.

The anti-connexin agent (e.g., an anti-connexin polynucleotide) may bepresent in a substantially isolated form. It will be understood that theproduct may be mixed with carriers or diluents that will not interferewith the intended purpose of the product and still be regarded assubstantially isolated. A product of the invention may also be in asubstantially purified form, in which case it will generally compriseabout 80%, 85%, or 90%, including, for example, at least about 95%, atleast about 98% or at least about 99% of the polynucleotide or dry massof the preparation.

Depending on the intended route of administration, the pharmaceuticalproducts, pharmaceutical compositions, combined preparations andmedicaments of the invention may, for example, take the form ofsolutions, suspensions, instillations, sprays, salves, creams, gels,foams, ointments, emulsions, lotions, paints, sustained releaseformulations, or powders, and typically contain about 0.01% to about 1%of active ingredient(s), about 1%-50% or active ingredient(s), about2%-60% of active ingredient(s), about 2%-70% of active ingredient(s), orup to about 90% of active ingredient(s). Other suitable formulationsinclude pluronic gel-based formulations,carboxymethylcellulose(CMC)-based formulations, andhyroxypropylmethylcellulose(HPMC)-based formulations. Other usefulformulations include slow or delayed release preparations.

Gels or jellies may be produced using a suitable gelling agentincluding, but not limited to, gelatin, tragacanth, or a cellulosederivative and may include glycerol as a humectant, emollient, andpreservative. Ointments are semi-solid preparations that consist of theactive ingredient incorporated into a fatty, waxy, or synthetic base.Examples of suitable creams include, but are not limited to,water-in-oil and oil-in-water emulsions. Water-in-oil creams may beformulated by using a suitable emulsifying agent with propertiessimilar, but not limited, to those of the fatty alcohols such as cetylalcohol or cetostearyl alcohol and to emulsifying wax. Oil-in-watercreams may be formulated using an emulsifying agent such as cetomacrogolemulsifying wax. Suitable properties include the ability to modify theviscosity of the emulsion and both physical and chemical stability overa wide range of pH. The water soluble or miscible cream base may containa preservative system and may also be buffered to maintain an acceptablephysiological pH.

Foam preparations may be formulated to be delivered from a pressurizedaerosol canister, via a suitable applicator, using inert propellants.Suitable excipients for the formulation of the foam base include, butare not limited to, propylene glycol, emulsifying wax, cetyl alcohol,and glyceryl stearate. Potential preservatives include methylparaben andpropylparaben.

Preferably the anti-connexin agents useful in practicing the instantinventions are combined with a pharmaceutically acceptable carrier ordiluent to produce a pharmaceutical composition. Suitable carriers anddiluents include isotonic saline solutions, for examplephosphate-buffered saline. Suitable diluents and excipients alsoinclude, for example, saline, dextrose, glycerol, or the like, andcombinations thereof. In addition, if desired substances such as wettingor emulsifying agents, stabilizing or ph buffering agents may also bepresent.

The term “pharmaceutically acceptable carrier” refers to anypharmaceutical carrier that does not itself induce the production ofantibodies harmful to the individual receiving the composition, andwhich can be administered without undue toxicity. Suitable carriers canbe large, slowly metabolized macromolecules such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, and amino acid copolymers.

Pharmaceutically acceptable salts can also be present, e.g., mineralacid salts such as hydrochlorides, hydrobromides, phosphates, sulfates,and the like; and the salts of organic acids such as acetates,propionates, malonates, benzoates, and the like.

The pharmaceutical carriers in the pharmaceutical composition useful inthe methods and articles of manufacture of this disclosure may be one ormore pharmaceutical carriers suitable for topical administration. In oneembodiment, the pharmaceutical carrier may be a nonionicpolyoxyethylene-polyoxypropylene copolymer, also referred to as apoloxamer. The pharmaceutical carrier may be present in thepharmaceutical composition at between 5 and 25-30%. For example, thepharmaceutical carrier may be present in the pharmaceutical compositionat 20% (w/w). In another embodiment the pharmaceutical carrier may bepresent in the pharmaceutical composition at about 22.0%. A preferredpoloxamer is poloxamer 407, also known as Pluronic F-127 (BASF)

The pharmaceutical compositions for use in the methods and kits andarticles of manufacture as disclosed herein may be formulated in adelayed release preparation, a slow release preparation, an extendedrelease preparation, a controlled release preparation, and/or in arepeat action preparation to a subject with a wound characterized inwhole or in part by delayed or incomplete wound healing, or other woundthat does not heal at an expected rate. Such formulations areparticularly advantageous for wounds that do not heal at expected rates,such as chronic wounds.

Suitable carrier materials include any carrier or vehicle commonly usedas a base for creams, lotions, sprays, foams, gels, emulsions, lotionsor paints for topical administration. Examples include emulsifyingagents, inert carriers including hydrocarbon bases, emulsifying bases,non-toxic solvents or water-soluble bases. Particularly suitableexamples include pluronics, HPMC, CMC and other cellulose-basedingredients, lanolin, hard paraffin, liquid paraffin, soft yellowparaffin or soft white paraffin, white beeswax, yellow beeswax,cetostearyl alcohol, cetyl alcohol, dimethicones, emulsifying waxes,isopropyl myristate, microcrystalline wax, oleyl alcohol and stearylalcohol.

Slow release gels in which the anti-connexin agent is released over timeare preferred for topical application. Thus, in preferred embodiments,the pharmaceutical composition may be formulated to provide sustainedrelease of the anti-connexin agent, e.g., an anti-connexin antisensepolynucleotide. Preferred anti-connexin polynucleotides includeanti-connexin 43 polynucleotides, particularly anti-connexin 43antisense polynucleotides.

Preferably, the pharmaceutically acceptable carrier or vehicle is a gel,suitably a nonionic polyoxyethylene-polyoxypropylene copolymer gel, forexample, a Pluronic gel, preferably Pluronic F-127 (BASF Corp.). Such agel can be used as a liquid at low temperatures but rapidly sets atphysiological temperatures, which can assist in confining the release ofthe anti-connexin agent, particularly an anti-connexin antisensepolynucleotide (e.g., an ODN) active ingredient, to the site ofapplication or immediately adjacent to that site.

Other pharmaceutically acceptable carriers useful in the articles ofmanufacture and methods of this disclosure include an alginate,polyvinyl alcohol, hydrogels, including hydrogels that contain acellulose derivative and/or polyacrylic acid; cellulose-based carrier,including hydroxyethyl cellulose, hydroxymethyl cellulose, carboxymethylcellulose, hydroxypropylmethyl cellulose and mixtures thereof.

Other suitable formulations include pluronic gel-based formulations,carboxymethylcellulose(CMC)-based formulations, andhydroxypropylmethylcellulose (HPMC)-based formulations. The compositionmay be formulated for any desired form of delivery, including topical,instillation, parenteral, subcutaneous, or transdermal administration.Other useful formulations include slow or delayed release preparations.

The formulation that is administered may contain transfection agents.Examples of such agents include cationic agents (for example calciumphosphate and DEAE-dextran) and lipofectants (for example Lipofectam™and Transfectam™), and surfactants.

In some embodiments, the formulation further includes a surfactant toassist with polynucleotide cell penetration or the formulation maycontain any suitable loading agent. Any suitable non-toxic surfactantmay be included, such as DMSO. Alternatively, a transdermal penetrationagent such as urea may be included.

In some embodiments, the effective dose for a given subject preferablylies within the dose that is therapeutically effective for at least 50%of the population, and that exhibits little or no toxicity at thislevel.

The effective dosage of each of the anti-connexin agents employed in themethods and compositions of the invention may vary depending on a numberof factors, including the particular anti-connexin agent(s) employed,the mode(s) of administration, the frequency of administration, thewound being treated, the severity of the wound being treated, the routeof administration, the needs of a patient sub-population to be treated,or the needs of the individual patient which different needs can be dueto age, sex, body weight, or relevant medical wound specific to thepatient.

A suitable dose may be from about 0.001 mg/kg to about 10 mg/kg bodyweight, such as about 0.01 mg/kg to about 0.1-1.0 mg/kg body weight. Asuitable dose may, however, be from about 0.001 mg/kg to about 0.1 mg/kgbody weight, such as about 0.01 mg/kg to about 0.050 mg/kg body weight.Doses from about 1 to 100, 200, 300, 400, and 500 micrograms (μg) can beused, with doses of about 30 μg to about 500 μg being preferred. Inother embodiments, the doses will be about 10.0, 11.0, 12.0, 13.0, 14.0,15.0, 16.0, 17.0, 18.0, 19.0, 20.0, 21.0, 22.0, 23.0, 24.0, 25.0, 26.0,27.0, 28.0, 29.0, 30.0, 31.0, 32.0, 33.0, 34.0, 35.0, 36.0, 37.0, 38.0,39.0, 40.0, 41.0, 42.0, 43.0, 44.0, 45.0, 46.0, 47.0, 48.0, 49.0, 50.0,52.5, 55.0, 57.5, 60.0, 62.5, 65.0, 67.5, 70.0, 72.5, 75.0, 77.5, 80.0,82.5, 85.0, 87.5, 90.0, 92.5, 95.0, 97.5, 100.0, 105, 110, 115, 120,125, 130, 135, 140, 145, 150, 155, 160, 65, 170, 175, 180, 185, 190,195, 200, 210, 220, 230, 250, 260, 270, 280, 290, 300, 310, 320, 330,340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470,480, 490, or about 500 milligrams per square centimeter, or any range orsubrange between any two of the recited doses, or any dose fallingwithin the range of about 1.0 to about 500 milligrams per squarecentimeter. As noted herein, repeat applications are contemplated.Repeat applications are typically applied about once per week, or whenwound-healing may appear to be stalled or slowing.

Still other useful dosage levels include those having between about 1nanogram (ng)/kg and about 1 mg/kg body weight per day of ananti-connexin agent described herein. In certain embodiments, the dosageof each of the subject compounds will generally be in the range of about1 ng/kg to about 1 μg/kg body weight, about 1 ng/kg to about 0.1 μg/kgbody weight, about 1 ng/kg to about 10 ng/kg body weight, about 10 ng/kgto about 0.1 μg/kg body weight, about 0.1 μg/kg to about 1 μg/kg bodyweight, about 20 ng/kg to about 100 ng/kg body weight, about 0.001 mg/kgto about 100 mg/kg body weight, about 0.01 mg/kg to about 10 mg/kg bodyweight, or about 0.1 mg/kg to about 1 mg/kg body weight. In certainembodiments, the dosage of an anti-connexin agent will generally be inthe range of about 0.001 mg/kg to about 0.01 mg/kg body weight, about0.01 mg/kg to about 0.1 mg/kg body weight, about 0.1 mg/kg to about 1mg/kg body weight, or about 1 mg/kg body weight. If more than oneanti-connexin agent is used, the dosage of each anti-connexin agent neednot be in the same range as the other. For example, the dosage of oneanti-connexin agent may be between about 0.01 mg/kg to about 1 mg/kgbody weight, and the dosage of another anti-connexin agent may bebetween about 0.1 mg/kg to about 1 mg/kg body weight. The dosage mayalso be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0,3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0,16.0, 17.0, 18.0, 19.0, 20.0, 21.0, 22.0, 23.0, 24.0, 25.0, 26.0, 27.0,28.0, 29.0, 30.0, 31.0, 32.0, 33.0, 34.0, 35.0, 36.0, 37.0, 38.0, 39.0,40.0, 41.0, 42.0, 43.0, 44.0, 45.0, 46.0, 47.0, 48.0, 49.0, 50.0, 52.5,55.0, 57.5, 60.0, 62.5, 65.0, 67.5, 70.0, 72.5, 75.0, 77.5, 80.0, 82.5,85.0, 87.5, 90.0, 92.5, 95.0, 97.5, or about 100.0 mg per kg bodyweight, or any range or subrange between any two of the recited doses,or any dose falling within the range of from about 0.1 to about 100 mgper kg body weight. As noted herein, repeat applications arecontemplated.

Other useful doses range from about 1 to about 10 μg per squarecentimeter (μg/cm²) of wound size. Certain doses will be about 1-2,about 1-5, about 2-4, about 5-7, and about 8-10 μg/cm² of wound size.Other useful doses are greater than about 10 μg/cm² of wound size,including about 15 μg/cm² of wound size, about 20 μg/cm² of wound size,about 25 μg/cm² of wound size, about 30 μg/cm² of wound size, about 35μg/cm² of wound size, about 40 μg/cm² of wound size, about 50 μg/cm² ofwound size, and about 100 μg/cm² of wound size. Other useful doses areabout 150 g/cm² of wound size, about 200 μg/cm² of wound size, about 250μg/cm² of wound size, or about 500 μg/cm² of wound size. As notedherein, repeat applications are contemplated.

For example, in certain embodiments, the anti-connexin agent compositionmay be applied at about 50 μM to about 5000 μM final concentration atthe treatment site and/or adjacent to the treatment site. Preferably,the anti-connexin agent composition is applied at about 100 μM to about3000 μM final concentration, more preferably, the anti-connexinpolynucleotide composition is applied at about 150 μM to about 3000 μMfinal concentration, and more preferably, the anti-connexinpolynucleotide composition is applied at about 150 μM to about 3300 μMfinal concentration. Additionally, the anti-connexin polynucleotidecomposition is applied at about 150 μM to about 3000 μM finalconcentration, and alternatively the anti-connexin polynucleotidecomposition is applied at about 250 μM to about 1000 μM finalconcentration, or at about 300 to about 1000 μM final concentration. Incertain other embodiments, the anti-connexin polynucleotide is appliedat about 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM,900 μM, 1000 μM, 1100 μM, 1200 μM, 1300 μM, 1400 μM, 1500 μM, 1600 μM,1700 μM, 1800 μM, 1900 μM, 2000 μM, 2100 μM, 2200 μM, 2300 μM, 2400 μM,2500 μM, 2600 μM, 2700 μM, 2800 μM, 2900 μM, or about 3000 μM finalconcentration, or any range in between any two of these concentrations.In other embodiments, the anti-connexin agent is applied at about a 20μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 10-200 μM,200-300 μM, 300-400 μM, 400-500 μM, 500-600 μM, 600-700 μM, 700-800 μM,800-900 μM, 900-1000 or 1000-1500 μM, or 1500 μM-2000 μM, 2000 μM-3000μM, 3000 μM-4000 μM, 4000 μM-5000 μM, 5000 μM-6000 μM, 6000 μM-7000 μM,7000 μM-8000 μM, 8000 μM-9000 μM, 9000 μM-10,000 μM, 10,000 μM-11,000μM, 11,000 μM-12,000 μM, 12,000 μM-13,000 μM, 13,000 μM-14,000 μM,14,000 μM-15,000 μM, 15,000 μM-20,000 μM, 20,000 μM-30,000 μM, 30,000μM-50,000 μM, or greater, or any range or subrange between any two ofthe recited doses, or any dose falling within the range of from about 20μM to about 50,000 μM.

Anti-connexin polynucleotide dose amounts include, for example, about0.1-1, 1-2, 2-3, 3-4, or 4-5 micrograms (μg), from about 5 to about 10g, from about 10 to about 15 μg, from about 15 to about 20 μg, fromabout 20 to about 30 μg, from about 30 to about 40 μg, from about 40 toabout 50 μg, from about 50 to about 75 μg, from about 75 to about 100μg, from about 100 μg to about 250 μg and from 250 μg to about 500 μg.Dose amounts from 0.5 to about 1.0 milligrams (mg) or more or alsoprovided, as noted above. Dose volumes will depend on the size of thesite to be treated, and may range, for example, from about 25-100microliter (μL) to about 100-200 μL, from about 200-500 μL to about500-1000 μL doses are also appropriate for larger treatment sites. Asnoted herein, repeat applications are contemplated.

Conveniently, the anti-connexin agent(s) is(are) administered in asufficient amount to downregulate expression of a connexin protein, ormodulate gap junction formation for at least about 0.5 to 1 hour, atleast about 1-2 hours, at least about 2-4 hours, at least about 4-6hours, at least about 6-8 hours, at least about 8-10 hours, at leastabout 12 hours, or at least about 24 hours post-administration.

The dosage of each of the anti-connexin agents in accordance with thesubject invention may also be determined by reference to theconcentration of the composition relative to the size, length, depth,area, or volume of the area to which it will be applied. For example, incertain topical and other applications, e.g., instillation, dosing ofthe pharmaceutical compositions may be calculated based on mass (e.g.micrograms) of or the concentration in a pharmaceutical composition(e.g. μg/μL) per length, depth, area, or volume of the area ofapplication.

The initial and any subsequent dosages administered will depend uponfactors noted herein. Depending on the oligonucleotide, the dosage andprotocol for administration will vary, and the dosage will also dependon the method of administration selected, for example, local or topicaladministration.

The doses may be administered in single or divided applications. Thedoses may be administered once, or application may be repeated.

One or more anti-connexin agents may be administered by the same ordifferent routes. The various agents of the invention can beadministered separately at different times during the course of therapy,or concurrently in divided or single combination forms.

Preferably one or more anti-connexin agents useful for wound healing aredelivered by topical administration (peripherally or directly to asite), including but not limited to topical administration using solidsupports (such as dressings and other matrices) and medicinalformulations (such as gels, mixtures, suspensions and ointments). Insome embodiments, the solid support comprises a biocompatible membraneor insertion into a treatment site. In another embodiment, the solidsupport comprises a dressing or matrix. In one embodiment of theinvention, the solid support composition may be a slow release solidsupport composition, in which the one or more anti-connexin agent(s)useful for wound healing is(are) dispersed in a slow release solidmatrix such as a matrix of alginate, collagen, or a syntheticbioabsorbable polymer. Preferably, the solid support composition issterile or low bio-burden. In one embodiment, a wash solution comprisingone or more anti-connexin polynucleotides can be used.

One or more doses may be administered to a subject having a refractorywound. In some embodiments, one or more doses of the pharmaceuticalcomposition comprising the may be administered at appropriate intervals.In some embodiments, the pharmaceutical composition may be administereddaily, two to six times per week, or weekly. For example, thecomposition comprising the anti-connexin agent (e.g., an anti-connexinpolynucleotide) can be administered, delivered, or otherwise exposed tothe wound to treated for an effective period of time, for example, atleast about 0.5 hours, about 1-2 hours, about 2-4 hours, about 4-6hours, about 6-8 hours, or for longer periods, e.g., up to 24 hours ormore. Exposures of about 1-2 hour, 2-3 hour, and 4-8 hour perapplication or delivery is presently preferred. Alternatively, theanti-connexin agent-containing compositions and formulations describedherein can be administered repeatedly, for example, once per week untilhealing is seen to be proceeding or is complete, as desired. Forexample, compositions of the invention may also be applied morefrequently, 2-3 times/week. They may also be applied weekly, biweekly,or monthly. Application once or twice per week is presently preferred.The frequency of administration and dose may change over the course oftreatment as the wound area and volume change. In addition, furtherapplication(s) can be made in the event wound healing once again becomesstalled or delayed.

While the delivery period will be dependent upon both the site at whichthe downregulation is to be induced and the therapeutic effect which isdesired, continuous or slow-release delivery for about 0.5 hours, about1-2 hours, about 2-4 hours, about 4-6 hours, about 6-8, or about 24hours or longer is provided. In accordance with the present invention,this may be achieved by inclusion of the anti-connexin agent(s) in aformulation together with a pharmaceutically acceptable carrier orvehicle, particularly in the form of a formulation for continuous orslow-release administration.

As noted, the one or more anti-connexin agents described may beadministered before, during, immediately following wounding, forexample, or within about 180 or more, about 120, about 90, about 60, orabout 30 days, of wounding, for example.

The routes of administration and dosages described herein are intendedonly as a guide since a skilled physician will determine the optimumroute of administration and dosage for any particular patient and wound.

Any of the methods of treating a subject having or suspected of havingor a disease, disorder, and/or wound, referenced or described herein mayutilize the administration of any of the doses, dosage forms,formulations, and/or compositions herein described.

Wound Treatment

In instances of tissue damage (particularly with wounds characterized bydelayed healing and chronic wounds) the formulations for use inaccordance with the invention have been found effective in bothpromoting the wound healing process, reducing inflammation and inminimizing scar tissue formation. The formulations therefore have clearbenefit in the treatment of wounds that do not heal at expected rates,whether the result of external trauma, or disease state (such asdiabetic ulcers) or condition (such as venous ulcers, arterial ulcers,and vasculitic ulcers) or physical processes (such as pressure ulcers).

In one aspect the invention is directed to a method of promoting orimproving wound healing in a subject suffering from or a chronic wound,delayed healing wound or incomplete healing wound, or other wounds thatdo not heal at expected rates, comprising administration of atherapeutically effective amount of one or more anti-connexin agents. Incertain embodiments, the administration of one or more anti-connexinagents is effective to reduce granulation tissue deposition, promotecell migration to accelerate wound closure and healing, to facilitateepithelial growth, or any combination thereof.

In one aspect the invention is directed to a method of promoting orimproving wound healing in a subject, comprising administration of oneor more anti-connexin agents in an amount effective to regulateepithelial basal cell division and growth in a chronic wound, delayedhealing wound or incomplete healing wound, or other wound that does notheal at an expected rate. In one embodiment, the anti-connexin agent isan anti-connexin antisense polynucleotide effective to regulateepithelial basal cell division and growth. In some embodiments, theanti-connexin antisense polynucleotide is an anti-connexin 26 antisensepolynucleotide, an anti-connexin 43 antisense polynucleotide, or amixture thereof.

In one aspect the invention is directed to a method of promoting orimproving wound healing, comprising administration of one or moreanti-connexin agents in an amount effective to regulate outer layerkeratin secretion in a chronic wound, delayed healing wound orincomplete healing wound, or other wound that does not heal at anexpected rate. In some embodiments, the anti-connexin agent is ananti-connexin antisense polynucleotide effective to regulate outer layerkeratin secretion. In one embodiment, the connexin antisensepolynucleotide is an anti-connexin 43 antisense polynucleotide, ananti-connexin 31.1 antisense polynucleotide, or a mixture thereof.

In one aspect the invention is directed to methods of reducing,preventing, or ameliorating tissue damage in a subject suffering from achronic wound, delayed healing wound or incomplete healing wound, orother wound that does not heal at an expected rate, comprisingadministration of one or more anti-connexin agents.

In one aspect the invention is directed to sustained administration ofone or more anti-connexin agents. In some embodiment, the anti-connexinagents are administered for at least about 1-24 hours, at least about0.5 hours, at least about 1 hour, at least about 2 hours, at least about3 hours, at least about 4 hours, at least about 5 hours, at least about6 hours, at least about 7 hours, at least about 8 hours, at least about9 hours, at least about 10 hours, at least about 11 hours, at leastabout 12 hours or at least about 24 hours. In some embodiments, connexinexpression is downregulated over a sustained period of time. Preferably,connexin 43 expression is downregulated for a sustained period of time.Conveniently, connexin 43 expression is downregulated for at least about0.5, 1, 2, 4, 6, 8, 10, 12, or 24 hours. Full recovery of connexinexpression generally occurs within at least about 48-72 hours followingdownregulation of expression. Suitable subjects for treatment inaccordance with the invention include diabetic subjects or othersubjects having a wound that does not heal at an expected rate.

In one aspect, the present invention provides methods of treating asubject having a chronic wound, delayed healing wound or incompletehealing wound, or other wound that does not heal at an expected rate,which comprises sustained administration of an effective amount of oneor more anti-connexin agents. In a further aspect, the present inventionprovides methods of promoting or improving wound healing in a subjectwhich comprises sustained administration of one or more anti-connexinagents to a chronic wound, delayed healing wound or incomplete healingwound, or other wound that does not heal at an expected rate.

According to another further aspect, the present invention providesmethods of promoting or improving wound healing in a subject having achronic wound, delayed healing wound or incomplete healing wound, orother wound that does not heal at an expected rate, which comprisessustained administration of one or more anti-connexin agents to a woundarea in an amount effective to increase re-epithlialization rates in thewound area. In some embodiments, such methods comprise sustainedadministration of an anti-connexin 43 antisense polynucleotide, and/oran anti-connexin 31.1 antisense polynucleotide. In some embodiments, thecomposition or compositions are administered in a sustained releaseformulation. In other embodiments, the composition or compositions areadministered for a sustained period of time. Conveniently, thecomposition is effective to decrease connexin 43 and/or 31.1 levels orexpression for at least about 24 hours. Subjects that may be treatedinclude diabetic subjects or other subjects having a wound that does notheal at an expected rate.

In yet another aspect, the present invention provides methods ofpromoting or improving wound healing in a subject having a chronicwound, delayed healing wound or incomplete healing wound, or other woundthat does not heal at an expected rate, which comprises sustainedadministration one or more anti-connexin agents to a wound area in anamount effective to effective to regulate epithelial basal cell divisionand growth and/or effective to regulate outer layer keratin secretion.In one embodiment, the composition comprises an anti-connexin antisensepolynucleotide effective to regulate epithelial basal cell division orgrowth, preferably an anti-connexin 26 antisense polynucleotide, ananti-connexin 43 antisense polynucleotide, anti-connexin 30 antisensepolynucleotide or a mixture thereof, for example. In some embodiments,the composition comprises an anti-connexin antisense polynucleotideeffective to regulate outer layer keratinization, preferably, ananti-connexin 31.1 antisense polynucleotide. In some embodiments, thecomposition or compositions are administered in a sustained releaseformulation. In other embodiments, the composition or compositions areadministered for a sustained period of time. Conveniently, thecomposition is effective to decrease connexin 43, 26, and/or 30 levelsor expression for at least about 24 hours. Subjects that may be treatedinclude diabetic subjects.

In one aspect the invention is directed to methods for treatment orprophylaxis of skin wounds, including a refractory chronic wound,refractory delayed healing wound or refractory incomplete healing wound,or other refractory wound that does not heal at an expected rate,comprising administering to a subject in need thereof an effectiveamount of an anti-connexin agent administered to said wound or a tissueassociated with said wound. In some embodiments, a composition of thepresent disclosure is administered to the skin or a tissue associatedwith the skin of said subject for an effective period of time.Conveniently, the composition is effective to decrease connexin 43levels, or block or reduce connexin 43 hemichannel opening, for at leastabout 0.5 hours, about 1-2 hours, about 2-4 hours, about 4-6 hours,about 4-8 hours, about 12 hours, about 18 hours, or about 24 hours. Achronic skin wound suitable for treatment may, for example, be selectedfrom the group consisting of pressure ulcers, diabetic ulcers, venousulcers, arterial ulcers, vasculitic ulcers, and mixed ulcers. Thechronic wound may be an arterial ulcer, which comprises ulcerationsresulting from complete or partial arterial blockage. The chronic woundmay be a venous stasis ulcer, which comprises ulcerations resulting froma malfunction of the venous valve and the associated vascular disease.The chronic wound may be a trauma-induced ulcer. Subjects with otherulcers may also be treated, including those with venous ulcers andothers described herein and known in the art.

Compositions

The present invention is directed to pharmaceutical compositions,formulations, and their methods of manufacture and use wherein suchcompositions comprise a therapeutically effective amount of ananti-connexin agent, including, for example, an anti-connexinpolynucleotide, including anti-connexin antisense polynucleotides. Thecompositions are useful in enhancing or promoting healing of wounds thatdo not heal at expected rates, including wounds that may be slow to healor refractory to conventional wound treatment or wound healing promotingtherapies.

In one preferred form, such compositions contain one or moreanti-connexin agent species, for example, an anti-connexin antisensepolynucleotide, to the mRNA or pre-mRNA of one connexin protein only.Most preferably, this connexin protein is connexin 43. Alternatively,the compositions may comprise agents, particularly polynucleotides, tomore than one connexin protein. Preferably, one of the connexin proteinsto which such agents are directed is connexin 43. Other connexinproteins to which anti-connexin agents may be directed include, forexample, connexins 26, 30, 30.3, 31, 31.1, 32, 37, 40, and 45. Suitableexemplary polynucleotides (and ODNs) directed to various connexins areset forth elsewhere herein.

Many aspects of the invention are described with reference toanti-connexin polynucleotides, particularly oligodeoxynucleotides.However, it is understood that other suitable polynucleotides (such asRNA polynucleotides) may be used in these aspects. Other anti-connexinoligonucleotides are RNAi, siRNA, and shRNA oligonucleotides.

Accordingly, in one aspect, the invention provides compositions for usein therapeutic treatment, which comprises at least one anti-connexinagent, preferably an anti-connexin 43 polynucleotide. In certainpreferred embodiments, such composition further comprise apharmaceutically acceptable carrier or vehicle.

Kits, Medicaments and Articles of Manufacturer

Optionally, one or more anti-connexin agents may also be used in themanufacture of the medicament. In one embodiment, the medicamentcomprises a therapeutically effective amount of an anti-connexin agent,preferably an anti-connexin 43 polynucleotide, and a pharmaceuticallyacceptable carrier.

As described, the kits and packages of the invention include an articleof manufacture comprising one or more containers or vessels thatcontains a sufficient amount of the desired anti-connexin agent(s) sothat a therapeutically effective amount of such agent(s) can bedelivered to a patient having a delayed healing or other chronic skinwound, for example, a chronic venous ulcer, venous stasis ulcer,arterial ulcer, pressure ulcer, diabetic ulcer, diabetic foot ulcer,vasculitic ulcer, decubitus ulcer, burn ulcer, trauma-induced ulcer,infectious ulcer, mixed ulcer, or pyoderma gangrenosum. Such kits alsoinclude instructions for the use of such agent(s). For example, suchinstructions may include directions regarding use for the treatment of asubject having a chronic wound or a wound characterized by delayedhealing. Instructions may include instructions for use with regard towounds that do not heal at expected rates, such as delayed healingwounds, incomplete healing wounds and chronic wounds. Such instructionsprovide directions regarding observing the patient to be treated in therun in period to determine if the wound is a refractory chronic wound,and, if so, directions regarding the administration of a therapeuticallyeffective amount of a composition comprising the anti-connexin agent inthe kit so as to effect treatment of the patient's wound.

In one aspect, the invention provides kits that comprise one or morecompositions or formulations described herein. For example, the kit mayinclude a composition comprising an effective amount of one or moreanti-connexin 43 antisense polynucleotides.

Articles of manufacture are also provided, comprising a vesselcontaining a composition or formulation for use in accordance with theinvention as described herein and instructions for use for the treatmentof a subject. For example, in preferred embodiments the article ofmanufacture comprises a vessel containing an anti-connexin agent andinstructions for use for the treatment of a subject suffering from achronic, delayed healing, or incomplete healing wound, or other woundthat does not heal at an expected rate.

The inventions also relate to the use of anti-connexin agents, such asanti-connexin polynucleotides, alone or in combination with transdermalpatches, dressings, bandages, matrices, and coverings capable of beingadhered or otherwise associated with the skin of a subject. As will beappreciated, such compositions and articles are capable of delivering atherapeutically effective amount of one or more anti-connexin agents,e.g., an anti-connexin polynucleotide such as an anti-Cx43 antisenseODN, to a delayed healing or chronic wound or the skin adjacent to sucha wound.

In another aspect, the invention includes articles of manufacturecomprising a vessel containing a therapeutically effective amount of oneor more anti-connexin agents and instructions for use, including use forthe treatment of a subject having a chronic wound or a delayed orincomplete healing wound, or other wound that does not heal at anexpected rate, or a disease, disorder, and/or condition characterized inwhole or in part by a chronic wound or delayed or incomplete woundhealing, or other wound that does not heal at an expected rate.

As noted, wound healing has been reported to be slow in diabetes, oftenresulting in infection or chronic wounds that can lead to amputation.Cell-cell communication through the gap junction protein connexin 43 andthe dynamic regulation of connexin 43 expression play pivotal roles inwound healing. In normal tissue, such as skin, in the first 24 hoursafter wounding, connexin 43 is normally downregulated and connexin 26upregulated in keratinocytes at the edge of the wound as they adopt amigratory phenotype. However, in diabetic tissue, in general, and skin,in particular, it has been found that connexin 43 is upregulatedimmediately after wounding.

The examples below describe the surprising and unexpected discovery thatwounds that remain within a certain size range (i.e., the “target sizerange”) during a pretreatment phase can be more effectively andefficaciously treated than wounds that more greatly increase or decreasein size during the pretreatment phase. In particular, target size rangechanges during the pretreatment phase that provide for effective therapysurprisingly and unexpectedly have been discovered to range from anincrease in wound size by not more than about 30% to a decrease in woundsize by not more than 35% during the pretreatment phase. Preferredpretreatment phases range from about 1 to about 30 days, preferably fromabout 5 days to about 20 days, even more preferably from about 7 days toabout 14 days.

Various aspects of the invention will now be described with reference tothe following examples, which will be understood to be provided by wayof illustration only and not to constitute a limitation on the scope ofthe invention.

EXAMPLES Example 1 Positive Phase 2 Efficacy of an Anti ConnexinFormulation in Chronic Venous Lea Ulcers

This example describes the results of CoDa Therapeutics' successfulPhase 2b human clinical trial. The Phase 2b Study was a randomized,parallel group, dose-ranging, controlled, double-blind Phase 2b clinicalstudy to evaluate the safety and efficacy of a pharmaceuticalformulation (Anti Connexin Formulation) comprising a single-strandedanti-connexin 43 oligo deoxyribonucleotide in 22.6% nonionicpolyloxyethylene-polyoxypropylene copolymer (topically applied topatients with venous leg ulcers (VLUs) over a 10-week treatment period.The active pharmaceutical ingredient was a Cx43asODN having thenucleotide sequence of SEQ ID NO: 1. The primary purpose of the studywas to determine if the can improve healing efficacy for subjects havinga VLU. Secondary objectives included determining whether high or lowdose Anti Connexin Formulation is safe and tolerable for VLU patients,to identify which Anti Connexin Formulation dose concentration is moreeffective in treating patients with VLUs, and to assess the safety andtolerability of the Anti Connexin Formulation vehicle alone (toascertain that vehicle alone has no negative effect on compressionbandaging, the standard of care (SOC)).

In the Phase 2b Study, 313 patients male and female patients aged 18years or older were enrolled at multiple sites, and randomized in a1:1:1:1 ratio into one of four treatment arms: low (1.0 mg/mL) or high(3.0 mg/mL) dose Anti Connexin Formulation treatment, includingcompression bandaging (standard-of-care (SOC)); vehicle alone, inaddition to SOC; or SOC alone. Once 33 subjects were randomized into theSOC-only arm, that group was closed to recruitment and thereaftersubjects were randomized on a 1:1:1 basis into the high or lose AntiConnexin Formulation or vehicle arms of the Study. Patients in the threetreatment arms receiving Anti Connexin Formulation or vehicle plus SOCreceived once-weekly applications over a 10-week treatment period oruntil the first assessment of 100% VLU re-epithelialization, whicheveroccurred first. Reference VLU (RVLU) complete closure was defined as100% re-epithelialization without drainage confirmed after two visits,14 days apart. For example, when a RVLU complete closure is firstobserved in a particular patient (i.e., the VLU exhibits 100%re-epithelialization), the RVLU must remain closed for at least another14 days in order for it to be confirmed as being completed closed.

The primary study endpoint was assessed as the percentage (%) change inRVLU surface area within a 10-week treatment period, as determined byphotographic planimetry. Secondary study endpoints were: the time toRVLU complete closure within the 10-week treatment period; incidence ofRVLU complete closure within the 10-week treatment period; incidence ofRVLU complete closure at each visit within the 10-week treatment period;percentage of RVLU surface area reduction (SAR) at each visit within the10-week treatment period; incidence of ulcer recurrence during thepost-treatment period; and pain in the RVLU at each visit within the10-week treatment period, each determined by investigator assessment,except for pain assessment, which was assessed by the particular patienton a categorical scale.

Safety assessment was based on the incidence of adverse events, asdetermined by investigator assessment; incidence of RVLU infection, asdetermined by investigator assessment; laboratory results; and physicalexaminations.

The Study was broken into three periods, a 2-week screening or “run-in”period (study visits S1, S2, and S3), a treatment period of up to 10weeks (study visits TO-TI0), and a post-treatment period of up to 12weeks (study visits P0 to P5). The screening period was designed todetermine whether subjects were eligible for the treatment phase of theStudy. During the 10-week treatment period, high and low dose AntiConnexin Formulation or vehicle plus SOC were applied weekly using dosevolumes of approximately 0.1 mL (100 uL) per 1.0 cm² of VLU surfacearea. For a given subject, treatment stopped at the first to occur of 10Anti Connexin Formulation or vehicle administrations or 100%re-epithelialization of the RVLU, at which point the subjecttransitioned to the post-treatment period. Subjects failing to achieve100% re-epithelialization of her/his RVLU at the T10 visit were exitedfrom the Study, and contacted 30 days thereafter to assess for seriousadverse events. The post-treatment period was intended to confirm RVLUcomplete closure, assess closure durability, and to continue to monitorfor serious adverse events within 30 days after cessation of treatment.If a patient's RVLU was not 100% closed by the P2 visit, s/he resumedtreatment. If a patient's RVLU was not 100% closed by the P3, P4, or P5visit, s/he was thereafter exited from the Study.

At of before the first screening period visit (S1), written informedconsent was obtained. At the S1 visit for each patient, a Studyinvestigator selected a single chronic RVLU, i.e., persisting for atleast 30 days prior to the S1 visit. Any RVLU clinical diagnosis wassupported by venous duplex ultrasonography demonstrating venous refluxof greater than 0.5 seconds. The RVLU had to be a full thickness,well-circumscribed lesion with defined boundaries. At the end of thescreening period (S3), the RVLU had to have a clean wound base free ofnon-viable tissue. Each RVLU had to be confirmed by review of woundphotographs by a medical monitor. Patients had to have an ankle brachialindex of greater than 0.80 measured at the S1 visit or within 3 monthsprior to the S1 visit. Any RVLU had to have a border of at least 1.5 cmof healthy skin between the outer edge of the wound bed and anysurrounding skin breakdown, wound, or ulcer after debridement at the endof the S3 visit. The RVLU had to have an estimated surface area ofbetween 2 cm² and 20 cm² at the end of the S3 visit, as determined byruler measurement (calculated using the longest length and longest widthperpendicular the longest length). Each patient had to be able totolerate high compression bandaging (˜40 mmHg at the ankle) and had tohave been compliant with standardized compression bandaging over thescreening period (visits S1-S3).

A subject was not eligible for enrollment if s/he had a decrease orincrease greater than 40% in the estimated RVLU surface area during the2-week screening period (to eliminate “fast healers” or those unlikelyto benefit from treatment, respectively; more than 75% of the RVLU wason or below the malleous; the RVLU had been treated with continuous highcompression for more than one year before the S1 visit; the RVLU woundbed had exposed bone, tendon, or fascia; the RVLU had clinical signs ofinfection and or biopsy proof of more than 10⁵ organisms per gram oftissue during the screening period; the subject had cellulitis in theRVLU leg during the screening period; the RVLU had a high volume ofexudate at the S1 visit that necessitates more than one high compressionbandage per week; or the subject has osteomyelitis. Other standardexclusion criteria also applied, including cancer, pregnancy orbreastfeeding, recent PDGF-BB, dermal substitute, or living skintreatment, being non-ambulatory, etc.

The results for Study are shown in FIGS. 1A-1C. These results show whenthe full Study cohort is considered, a higher than expected responserate in the vehicle-alone arm was observed. Indeed, the results from thevehicle-alone plus SOC subjects showed more complete wound closures andgreater RVLU surface are reductions than observed for the low dose AntiConnexin Formulation arm. Re-analysis of the data unexpectedly revealed,in contrast to FDA guidance, that an increase or decrease of RVLU SAR of+40% to −40% surprisingly led to “rapid healers” (i.e., those who healwithout the need for pharmaceutical intervention) being recruited intothe study population. When a different range of RVLU SAR increase ordecrease was utilized, namely −15% to +30%, a 178-patient subpopulationwas identified that exhibited statistically significant (p<0.05)responses in the context of the primary SAR endpoint and the completewound closure secondary endpoint. These results are shown in FIGS. 2, 3,and 4. This demonstrates that patients with refractory chronic woundshad statistically significant responses to treatment with the AntiConnexin Formulation. Similar efficacy responses were seen with woundsthat did not decrease in size by more than about 30% (+30%) over astandard-of-care treatment period using compression bandaging over twoweeks, and in wounds that did not decrease in size by more than about35% (+35%) over the two-week compression period, in wounds that did notincrease or decrease in size by more than about −50/+30%, −100%/+30%,−15%/+30%, −20%/+30%, 25%/+30% or −30%/+30% over the two-weekstandard-of-care treatment period using compression bandaging.

Together, the results indicate that selecting patients for treatmentwith Anti Connexin Formulation benefit from pre-screening prospectivepatients during a pretreatment or “run-in” phase to identify patientswhose chronic wounds, in this case VLUs, with surface areas that remainwithin a pre-determined target size range, preferably having a decreaseor increase in surface are during the “run-in” period of −15% to +30%,for example. Other ranges are described herein and above. Patientshaving chronic wounds, e.g., VLUs, that do not more greatly increaseand/or decrease in size during the “run-in” phase, from two to fourweeks, when then administered an anti-connexin composition, e.g., AntiConnexin 43 Formulation in this case, can be expected to favorablyrespond to treatment, for example, by experiencing complete woundclosure and/or by experiencing greater degrees of wound closure thanwould be otherwise be expected.

All patents, publications, scientific articles, web sites, and otherdocuments and materials referenced or mentioned herein are indicative ofthe levels of skill of those skilled in the art to which the inventionpertains, and each such referenced document and material is herebyincorporated by reference to the same extent as if it had beenincorporated by reference in its entirety individually or set forthherein in its entirety. The inventors (or their assignee(s)) reserve theright to physically incorporate into this specification any and allmaterials and information from any such patents, publications,scientific articles, web sites, electronically available information,and other referenced materials or documents.

The specific methods and compositions described herein arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects, and embodiments will occur to those skilled in the art uponconsideration of this specification, and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, which is notspecifically disclosed herein as essential. Thus, for example, in eachinstance herein, in embodiments or examples of the present invention,any of the terms “comprising”, “consisting essentially of”, and“consisting of” may be replaced with either of the other two terms inthe specification. Also, the terms “comprising”, “including”,“containing”, etc. are to be read expansively and without limitation.

The methods and processes illustratively described herein suitably maybe practiced in differing orders of steps, and that they are notnecessarily restricted to the orders of steps indicated herein or in theclaims. It is also understood that as used herein and in the appendedclaims, the singular forms “a,” “an,” and “the” include plural referenceunless the context clearly dictates otherwise. Under no circumstancesmay the patent be interpreted to be limited to the specific examples orembodiments or methods specifically disclosed herein. Under nocircumstances may the patent be interpreted to be limited by anystatement made by any Examiner or any other official or employee of thePatent and Trademark Office unless such statement is specifically andwithout qualification or reservation expressly adopted in a responsivewriting by or on behalf of the inventors.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intent in the use ofsuch terms and expressions to exclude any equivalent of the featuresshown and described or portions thereof, but it is recognized thatvarious modifications are possible within the scope of the invention asclaimed. Thus, it will be understood that although the present inventionhas been specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

1-47. (canceled)
 48. A method of treating a refractory wound,comprising: a. measuring the size a skin wound upon initial presentationfor treatment to obtain a first size measurement; b. administeringstandard of care such as compression bandaging and/or off-loading to thewound; c. measuring the size of the wound after 2-4 weeks ofadministering the standard of care to obtain a second size measurement;d. determining that the second size indicator of the wound is within apredetermined range from about −30 to about +35% of the first sizemeasurement, thereby identifying a refractory wound; and e.administering to the refractory wound a pharmaceutical compositioncomprising a pharmaceutical composition comprising a pharmaceuticalcarrier suitable for topical administration of an anti-connexinpolynucleotide to a connexin selected from connexin 26, connexin 30 andconnexin
 43. 49. A method of treating a refractory venous leg ulcer,comprising administering to the ulcer a pharmaceutical carrier suitablefor topical administration and about 3-30 mg/mL of an anti-connexinpolynucleotide to a connexin selected from connexin 26, connexin 30 andconnexin
 43. 50. An article of manufacture for use in treating arefractory wound, comprising a receptacle containing a compositioncomprising a pharmaceutical carrier suitable for topical administrationhaving about 20-23% of a nonionic polyoxyethylene-polyoxypropylene andabout 3-30 mg/mL of an anti-connexin oligodeoxynucleotide to a connexinselected from connexin 26, connexin 30 and connexin 43, and, andinstructions for the treatment of the refractory wound, by topicallyadministering the composition to or in the proximity of the wound.
 51. Amethod of detecting a refractory wound with an increased likelihood ofcomplete closure following topical administration to the recalcitrantwound of a composition comprising a pharmaceutical carrier suitable fortopical administration of an anti-connexin oligodeoxynucleotide to aconnexin selected from connexin 26, connexin 30 and connexin 43, themethod comprising: a. measuring the size of a skin wound upon initialpresentation for treatment, to obtain a first size measurement; b.administering standard of care, such as compression bandaging and/oroff-loading to the wound; c. measuring the size of the skin wound afterabout 2-4 weeks of administering the standard of care, to obtain asecond size measurement; d. detecting that the second size measurementis within −30% to +35% of the first size measurement, thereby detectinga refractory wound having an increased likelihood of complete closurefollowing topical administration to the recalcitrant wound of apharmaceutical composition comprising a pharmaceutical carrier suitablefor topical administration having about 20-23% of a nonionicpolyoxyethylene-polyoxypropylene and about 3-30 mg/mL of ananti-connexin oligodeoxynucleotide to a connexin selected from connexin26, connexin 30 and connexin 43; and e. administering the pharmaceuticalcomposition to the wound.
 52. The method of claim 48, wherein measuringthe first or second size indicator further comprises using planimetry,digitizing techniques, stereophotogrammetry, a ruler, wound tracing, ahandheld laser scanner or a camera.
 53. A method according to claim 52,wherein the refractory wound is selected from the group consisting of avenous ulcers, venous stasis ulcers, arterial ulcers, pressure ulcers,diabetic ulcers, diabetic foot ulcers, vasculitic ulcers, decubitusulcers, burn ulcers, trauma-induced ulcers, infectious ulcers, mixedulcers, and pyoderma gangrenosum.
 54. A method according to claim 48,wherein the chronic wound is a venous leg ulcer.
 55. A method accordingto claim 48, comprising a plurality of administrations of thepharmaceutical composition.
 56. A method according to claim 55, whereinthe pharmaceutical composition is applied repeatedly until wound closureis achieved.
 57. A method according to claim 56, wherein theadministrations are periodic.
 58. A method according to claim 57,wherein the periodic administrations occur at regularly scheduledintervals, optionally, daily, every other day, twice weekly, weekly,twice monthly, and monthly.
 59. A method according to claim 48, whereinthe anti-connexin 43 agent is an anti-connexin 43 polynucleotide.
 60. Amethod according to claim 48, wherein the anti-connexin 43polynucleotide, optionally an oligodeoxynucleotide or modifiedoligodeoxynucleotide comprises from about 18 to about 32 nucleotides.61. A method according to claim 48, wherein the subject is a mammal,optionally a human.
 62. The method of claim 48, wherein thepharmaceutical composition comprises a pharmaceutically acceptablecarrier and an anti-connexin 43 polynucleotide present at aconcentration selected from the following: from about 10-200 μM, 200-300μM, 300-400 μM, 400-500 μM, 500-600 μM, 600-700 μM, 700-800 μM, 800-900μM, 900-1000 or 1000-1500 μM, or 1500 μM-2000 μM, 2000 μM-3000 μM, 3000μM-4000 μM, 4000 μM-5000 μM, 5000 μM-6000 μM, 6000 μM-7000 μM, 7000μM-8000 μM, 8000 μM-9000 μM, 9000 μM-10,000 μM, 10,000 μM-11,000 μM,11,000 μM-12,000 μM, 12,000 μM-13,000 μM, 13,000 μM-14,000 μM, 14,000μM-15,000 μM, 15,000 μM-20,000 μM, 20,000 μM-30,000 μM, or from about30,000 μM-50,000 μM.
 63. The method according to claim 59, wherein theanti-connexin 43 polynucleotide is selected from: anoligodeoxynucleotide, a modified oligodeoxynucleotide, an unmodifiedoligodeoxynucleotide, an antisense polynucleotide, an unmodifiedantisense polynucleotide, and a modified antisense polynucleotide. 64.The method of claim 59, wherein the anti-connexin 43 polynucleotide is asequence selected from SEQ ID NOS: 1-3, or a sequence having up to about100 nucleotides of a sequence complementary to SEQ.ID.NO. 134 orSEQ.ID.NO.
 135. 65. The method of claim 59, wherein the anti-connexin 43polynucleotide is a sequence selected from SEQ ID NOS: 1 and
 2. 66. Themethod of claim 59, wherein the anti-connexin 43 polynucleotide is anantisense polynucleotide having at least about 70 percent homology withSEQ ID NOS: 1 and
 2. 67. The method of claim 59, wherein theanti-connexin 43 polynucleotide is an antisense polynucleotide thathybridizes to connexin 43 mRNA under conditions of medium to highstringency.
 68. The method according to claim 67, wherein said antisensepolynucleotide has a sequence selected from SEQ.ID.NO:1-3, or a sequencehaving up to about 40 nucleotides of a sequence complementary toSEQ.ID.NO. 134 or SEQ.ID.NO.
 135. 69. The method according to claim 67,wherein said antisense polynucleotide is selected from: 1.(SEQ ID NO: 1) GTA ATT GCG GCA AGA AGA ATT GTT TCT GTC; and 2.(SEQ ID NO: 2) GTA ATT GCG GCA GGA GGA ATT GTT TCT GTC
 3. (SEQ ID NO: 3)GGC AAG AGA CAC CAA AGA CAC TAC CAG CAT.


70. The method according to claim 67, wherein said antisensepolynucleotide has from about 15 to about 35 nucleotides and issufficiently complementary to connexin 43 mRNA to form a duplex having amelting point greater than 20° C. under physiological conditions. 71.The method according to claim 67, wherein the antisense polynucleotidehas from about 15 to about 35 nucleotides and has at least about 70percent homology to an antisense sequence of connexin 43 mRNA.
 72. Themethod according to claim 59, wherein said anti-connexin agent is anRNAi or siRNA polynucleotide.
 73. The method according to claim 48,which is formulated as a gel.
 74. The method according to claim 59,wherein the pharmaceutical formulation is administered topically. 75.The method according to claim 73, wherein said gel is apolyoxyethylene-polyoxypropylene copolymer-based gel or acarboxymethylcellulose-based gel.
 76. The method according to claim 75,wherein said gel is a pluronic gel.
 77. The method according to claim76, wherein said gel is a pluronic F-127.
 78. The method according toclaim 48, wherein the pharmaceutically acceptable carrier comprises analginate.
 79. The method according to claim 59, wherein thepharmaceutically acceptable carrier comprises a hydrogel.
 80. The methodaccording to claim 79, wherein the hydrogel comprises a hydrogelselected from the group consisting of hydrogels containing a cellulosederivative and hydrogels containing polyacrylic acid.
 81. The methodaccording to claim 48, wherein the pharmaceutically acceptable carrieris a cellulose-based carrier.
 82. The method according to claim 81,wherein the pharmaceutically acceptable carrier comprises acellulose-based carrier selected from the group consisting ofhydroxyethyl cellulose, hydroxymethyl cellulose, carboxymethylcellulose, hydroxypropylmethyl cellulose and mixtures thereof.
 83. Themethod according to claim 48, wherein the composition is formulated forsustained release.
 84. The method according to claim 48, wherein thecomposition is formulated for slow release, extended release, orcontrolled release.
 85. The method according to claim 48, wherein thecomposition is a cream, ointment, emulsion, lotion, spray, salve, foamor paint.
 86. A kit comprising package material containing a compositionfor use in the method of claim 59 together with instructions for use intreating a refractory chronic wound.
 87. A kit according to claim 86,wherein the wound is characterized at least in part by increasedexpression of connexin
 43. 88. A kit according to claim 86, wherein thewound is characterized at least in part by inflammation.
 89. A kitaccording to claim 86, wherein the wound is a dehiscent wound.
 90. A kitaccording to claim 86, wherein the wound is a venous ulcer.
 91. A kitaccording to claim 86, wherein the wound is a diabetic ulcer.
 92. A kitaccording to claim 86, wherein the wound is a diabetic foot ulcer.
 93. Akit according to claim 86, wherein the wound is a pressure ulcer.
 94. Akit according to claim 86, wherein the wound is an arterial ulcer.
 95. Akit according to claim 86, wherein the wound is a vasculitic ulcer. 96.A kit according to claim 86, wherein the wound is a skin ulcer resultingfrom trauma or a burn.
 97. A kit according to claim 86, wherein thesubject is diabetic.
 98. A kit according to claim 86, wherein thesubject has a venous valve malfunction and/or venous insufficiency. 99.A kit according to claim 86, wherein the subject has an arterialblockage.
 100. A kit according to claim 86, wherein said composition isapplied more than once.
 101. A kit according to claim 86, wherein theinstructions provide for administration of said composition about onceper week.
 102. A kit according to claim 86, wherein the instructionsprovide for bi-weekly administration of said composition.
 103. A kitaccording to claim 86, wherein the instructions provide foradministration of said composition every 3-7 days.
 104. A kit accordingto claim 86, wherein the subject is a human.
 105. A kit according toclaim 86, wherein the subject is a non-human animal.
 106. A method oftreating a subject having a wound not healing at an expected rate,comprising administering to the wound a composition comprising ananti-connexin 43 polynucleotide and a pharmaceutically acceptablecarrier, wherein the amount of said anti-connexin 43 polynucleotideadministered to said wound ranges from about 30 to about 500 μg persquare centimeter of said wound, the improvement comprising determiningwhether the wound heals by more than about 30-35% during a 2-4 weekrun-in period during which the wound is treated by compression and, ifnot, applying said anti-connexin 43 composition to said wound.